Methods and compositions for dosing in adoptive cell therapy

ABSTRACT

Provided are methods for administering multiple doses of cells, such as T cells, to subjects for cell therapy. Also provided are compositions and articles of manufacture for use in the methods. The cells generally express recombinant receptors such as chimeric receptors, e.g., chimeric antigen receptors (CARs) or other transgenic receptors such as T cell receptors (TCRs). The methods generally involve administering a first and at least one consecutive dose of the cells. Timing of the doses relative to one another, and/or size of the doses, in some embodiments provide various advantages such as lower or reduced toxicity and improved efficacy, for example, due to increased exposure of the subject to the administered cells. In some embodiments, the first dose is a relatively low dose, such as one that reduces tumor or disease burden, thereby improving the efficacy of consecutive or subsequent doses, and the consecutive dose is a consolidating dose.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application No.62/066,279 filed Oct. 20, 2014, entitled “Methods and Compositions forDosing in Adoptive Cell Therapy,” U.S. provisional application No.62/162,647, filed May 15, 2015, entitled “Methods and Compositions forDosing in Adoptive Cell Therapy,” U.S. provisional application No.62/168,710, filed May 29, 2015, entitled “Methods and Compositions forDosing in Adoptive Cell Therapy,” and U.S. provisional application No.62/215,732, filed Sep. 28, 2015, entitled “Methods and Compositions forDosing in Adoptive Cell Therapy,” the contents of which are incorporatedby reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled735042001000seqlist.txt, created Oct. 20, 2015, which is 16 kilobytes insize. The information in electronic format of the Sequence Listing isincorporated by reference in its entirety.

FIELD

The present disclosure relates to adoptive cell therapy involving theadministration of multiple doses of cells, and methods, compositions,and articles of manufacture for use in the same. The cells generallyexpress recombinant receptors such as chimeric receptors, e.g., chimericantigen receptors (CARs) or other transgenic receptors such as T cellreceptors (TCRs). Features of the methods, including the timing of thedoses and numbers of cells administered, provide various advantages,such as lower toxicity and/or improved efficacy, for example, due toincreased exposure of the subject to the administered cells. In someembodiments, the first dose involves a relatively lower number of cellscompared with dosage amounts administered in other methods. In someembodiments, the first dose reduces tumor or disease burden, therebyimproving the efficacy of consecutive or subsequent doses. In someembodiments, the timing of a consecutive dose is designed to minimizerisk of toxicity and/or host immune response to the cells by thesubject, thereby improving persistence and efficacy.

BACKGROUND

Various methods are available for adoptive cell therapy using engineeredcells expressing recombinant receptors, such as chimeric antigenreceptor (CARs). Improved methods are needed, for example, to reduce therisk of toxicity and/or to increase efficacy, for example, by increasingexposure of the subject to the administered cells, for example, byimproving expansion and/or persistence of the administered cells.Provided are methods, compositions, and articles of manufacture thatmeet such needs.

SUMMARY

Provided are methods of for administering to subjects cells expressinggenetically engineered (recombinant) cell surface receptors in adoptivecell therapy, for example, to treat diseases and/or conditions in thesubjects. The methods generally involve administering multiple doses ofsuch cells, and/or administering a consecutive dose to a subject havingbeen previously treated with a prior (e.g., first) dose of such cells.In some embodiments, the doses are a first dose and one or moreconsecutive doses. In some embodiments, the first dose is a relativelylow dose and/or is a conditioning or debulking dose and/or theconsecutive dose(s) is a consolidating dose(s). Also provided are cells,compositions, and articles of manufacture for use in such methods. Insome embodiments, the recombinant receptors are genetically engineeredantigen receptors, such as functional non-TCR antigen receptors, e.g.,chimeric antigen receptors (CARs) and other recombinant antigenreceptors such as transgenic T cell receptors (TCRs). Also among thereceptors are other recombinant chimeric receptors, such as thosecontaining an extracellular portion that specifically binds to a ligandor receptor or other binding partner and an intracellular signalingportion, such as the intracellular signaling portion of a CAR. The dosesin some embodiments include a relatively low first dose.

In some embodiments, the methods involve (a) administering to a subjectwith a disease or condition a first dose of cells expressing arecombinant receptor (e.g., chimeric antigen receptor (CAR)), the firstdose containing the cells; and (b) administering to the subject aconsecutive dose of recombinant receptor-expressing (e.g.,CAR-expressing) cells. In other embodiments, e.g., for providingconsolidating treatment, the methods are carried out by administering tothe subject the consecutive dose or doses as in (b), to a subject thathas been previously administered the first dose as in (a).

In some embodiments, the methods involve (a) administering to a subjecthaving a disease or condition a first dose of cells expressing arecombinant receptor (e.g., chimeric antigen receptor (CAR)). In someembodiments, the first dose contains no more than about 1×10⁶ of thecells per kilogram body weight of the subject, no more than about 1×10⁸of the cells, and/or no more than about 1×10⁸ of the cells/m² of thesubject. In some embodiments, the methods further involve (b)administering to the subject a consecutive dose of cells expressing arecombinant receptor (e.g., CAR) at a time point that is at least ormore than about 14 days after and less than about 28 days afterinitiation of the administration in (a).

In some embodiments, at the time of the administration in (b) the serumlevel in the subject of a factor indicative of cytokine release syndrome(CRS) is less than about 10 times, less than about 25 times, and/or lessthan about 50 times that in the subject immediately prior to saidadministration in (a). In some embodiments, the subject does not exhibitgrade 3 or higher neurotoxicity. In some embodiments, a CRS-relatedoutcome or symptom of neurotoxicity in the subject following theadministration of the first dose has reached a peak level and begun todecline following the administration in (a). In some embodiments, thesubject does not exhibit a detectable humoral or cell-mediated immuneresponse against the receptor (e.g., CAR) expressed by the cells of saidfirst dose.

In some embodiments, the methods further involve the administration ofadditional consecutive or subsequent doses, such that a first andmultiple consecutive doses are administered, e.g., in accordance withthe dosing amounts and timing schedules as specified for the first andconsecutive doses. In some embodiments, the first of one or moresubsequent doses is administered at a time that is at least or greaterthan 14 days after the initiation of the administration of theconsecutive dose. In some embodiments, the administration of the first,consecutive, and subsequent doses includes administering at least threeof the doses within at or about 28 days. In some embodiments, theconsecutive dose is administered at about day 14 following theinitiation of administration of the first dose, and an additionalconsecutive or subsequent dose is administered at day 28 following theinitiation of administration of the first dose. In some embodiments,additional subsequent doses are administered at day 42 and/or day 56following the initiation of administration of the first dose.

In some embodiments, the first dose is administered in an amountsufficient to reduce burden of the disease or condition in the subject.In some embodiments, the first dose is a low dose, such as a debulkingdose, such as a dose that is lower than that required to eradicate thedisease or condition but that may effect a reduction in burden or bulkof such disease or condition. In some embodiments, the administration ofthe first dose does not induce severe cytokine release syndrome (CRS) inthe subject. In some embodiments, administration of the first dose doesnot induce CRS in the subject. In some embodiments, based on clinicaldata, administration of the first dose does not induce severe CRS in amajority of subjects. In some embodiments, the administration of thefirst dose does not induce CRS encompassing a combination of (1)persistent fever (fever of at least 38 degrees Celsius for at leastthree days) and (2) a serum level of C reactive protein (CRP) of atleast at or about 20 mg/dL, and/or does not induce CRS encompassinghypotension requiring the use of two or more vasopressors or respiratoryfailure requiring mechanical ventilation.

In some embodiments, administration of the first dose does not inducegrade 3 or higher neurotoxicity in the subject. In some embodiments,based on clinical data, administration of the first dose does not inducegrade 3 or higher neurotoxicity in a majority of subjects. In someembodiments, symptoms associated with a clinical risk of neurotoxicityand/or grade 3 or higher neurotoxicity include confusion, delirium,expressive aphasia, obtundation, myoclonus, lethargy, altered mentalstatus, convulsions, seizure-like activity, seizures (optionally asconfirmed by electroencephalogram [EEG]), elevated levels of betaamyloid (Aβ), elevated levels of glutamate, and elevated levels ofoxygen radicals.

In some embodiments, the first dose is lower than a dose that wouldcause CRS or severe CRS in the subject. In some embodiments, the firstdose comprises no more than about 1×10⁶ of the cells per kilogram bodyweight of the subject, no more than 5×10⁵ of the cells per kilogram bodyweight of the subject, no more than about 1×10⁸ of the cells, or no morethan about 1×10⁸ of the cells/m² of the subject.

In some embodiments, the consecutive dose(s) is administered at a timeat which a clinical risk for neurotoxicity, cytokine-release syndrome(CRS), macrophage activation syndrome, or tumor lysis syndrome, is notpresent or has passed or has subsided following the administration ofthe first (or prior) dose. In some embodiments, the consecutive dose isadministered at a time at which a biochemical readout evidencing CRS,neurotoxicity, macrophage activation syndrome, or tumor lysis syndrome,is not present or has passed or has subsided following saidadministration of the first (or prior) dose. In some embodiments, theconsecutive dose(s) is administered at a time at which a serum level ofa factor indicative of cytokine-release syndrome (CRS) or neurotoxicityin the subject is less than about 10 times, less than about 25 times,and/or less than about 50 times the serum level of the indicator in thesubject immediately prior to said administration of the first dose. Insome embodiments, the consecutive dose(s) is administered at a timeafter a neurotoxicity and/or CRS-related outcome or serum factorindicating CRS in the subject has reached a peak level and begun todecline following said administration, such as where at the time of theadministration of the consecutive dose, the level of a CRS-relatedoutcome or serum factor indicative of CRS is no more than 50% of thepeak level, is no more than 20% of the peak level, or is no more than 5%of the peak level, or is at or about the level immediately prior to theadministration of the first dose.

In some embodiments, the subject does not exhibit cytokine releasesyndrome (CRS), does not exhibit severe CRS, does not exhibitneurotoxicity, does not exhibit severe neurotoxicity, or does notexhibit neurotoxicity above grade 3 following administration of thefirst dose and/or following the administration of the consecutive dose.

Among the CRS-related outcomes are fever, hypotension, hypoxia,neurologic disturbances, or a serum level of an inflammatory cytokines,such as interferon gamma (IFNγ), granulocyte-macrophagecolony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFα),IL-6, IL-10, IL-1β, IL-8, IL-2, MIP-1, Flt-3L, fracktalkine, and IL-5,and C reactive protein (CRP). Among the factors, e.g., serum factors,indicative of CRS are inflammatory cytokines such as IFNγ, GM-CSF, TNFα,IL-6, IL-10, IL-1β, IL-8, IL-2, MIP-1, Flt-3L, fracktalkine, and IL-5,and CRP.

In some aspects, the time of administering the consecutive dose(s) isfurther one at which the subject does not exhibit an immune response,e.g., does not exhibit a detectable adaptive host immune responsespecific for the receptor (e.g., CAR) expressed by the cells of saidfirst (or prior) dose.

In some embodiments, the time between the administration of the firstdose, e.g., the initiation of the administration of the first or priordose, and the initiation of the administration of the consecutive dose(e.g., the initiation of the administration of the consecutive dose) isgreater than about 4 days, e.g., greater than about 5, 6, 7, 8, or 9days, e.g., greater than about 20 days, e.g., between about 9 and about35 days, between about 14 and about 28 days, between 15 and 27 days, orbetween 17 days and about 21 days; and/or at or about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, or 27 days. In some embodiments,administration of the consecutive dose (e.g., initiation thereof) ismore than about 14 days after and less than about 28 days afteradministration of the first or prior dose (e.g., initiation thereof). Insome embodiments, the administration of the consecutive dose isinitiated 21 days following the initiation of the first dose. In someembodiments, the time between administration of the first and theconsecutive dose (e.g., initiation thereof) or prior and nextconsecutive dose is greater than about 14 days and less than about 28days, such as between 15 and 27 days, such as about 21 days. In someembodiments, the time between administration of the first and theconsecutive dose (e.g., initiation thereof) is about 17 days.

In some embodiments, at the time of the administration of theconsecutive dose, the serum level in the subject of a factor indicativeof CRS is less than about 10 times, less than about 25 times, and/orless than about 50 times that in the subject immediately prior to saidadministration of the first dose; and/or a CRS-related outcome in thesubject following said administration of said first dose has reached apeak level and begun to decline following the administration in (a);and/or the subject does not exhibit a detectable humoral orcell-mediated immune response against the receptor (e.g., CAR) expressedby the cells of said first dose. In some embodiments, at the time of theadministration in (b) or of the consecutive dose, the serum level ofsaid factor indicative of CRS is no more than ten times the levelimmediately prior to the administration in (a) or of the first dose.

In some embodiments, the subject has not received a dose of cellsexpressing the receptor (e.g., CAR) that is expressed by the cells inthe first dose prior to the administration in (a). In some embodiments,the receptor (e.g., the CAR) expressed by the cells in the consecutivedose contains at least one immunoreactive epitope present in thereceptor (e.g., CAR) expressed by the cells in the first dose. In someembodiments, the receptor in the cells of the consecutive dose isidentical or substantially identical to the receptor (e.g. the CAR)expressed by the cells in the first dose. In some embodiments, thereceptor expressed by the cells of the first dose specifically binds toan antigen expressed by a cell or tissue of the disease or condition orassociated with the disease or condition. In some embodiments, thereceptor expressed by the cells of the consecutive dose binds to thesame antigen, e.g., to the same epitope, and/or contains the sameantigen-binding domain as that in the first dose.

In some embodiments, the disease or condition is a tumor. In someembodiments, it is a cancer, malignancy, neoplasm, or otherproliferative disease or disorder, such as leukemia, lymphoma, e.g.,chronic lymphocytic leukemia (CLL), ALL, non-Hodgkin's lymphoma, acutemyeloid leukemia, multiple myeloma, refractory follicular lymphoma,mantle cell lymphoma, indolent B cell lymphoma, B cell malignancies,cancers of the colon, lung, liver, breast, prostate, ovarian, skin,melanoma, bone, and brain cancer, ovarian cancer, epithelial cancers,renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma,cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma,Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and/ormesothelioma. In some embodiments, the disease or condition is aleukemia or lymphoma. In some embodiments, the disease or condition isacute lymphoblastic leukemia. In some embodiments, the disease orcondition is non-Hodgkin lymphoma (NHL).

In some embodiments, the disease is a cancer and the subject does notexhibit morphologic disease at the time of initiation of theadministration of the consecutive dose. In some embodiments, the diseaseis a leukemia or lymphoma and the subject does not exhibit greater than5% blast cells in the bone marrow at the time of the administration ofthe consecutive dose. In some embodiments, the subject exhibitsdetectable molecular disease and/or minimum residual disease at the timeof the administration of the consecutive dose.

In some embodiments, administration of the first dose leads to areduction in burden of the disease or condition in the subject, forexample, as indicated by a reduction in one or more factors indicativeof disease burden following said administration of the first dose, e.g.,following the administration in (a). In some embodiments, at the time ofthe administration in (b) or of the consecutive dose, the subject hasnot relapsed and/or the one or more factors indicative of disease burdenhave not increased following the initial reduction experienced after thefirst dose. In some embodiments, the consecutive dose of cells containscells in an amount sufficient for reduction in burden of a disease orcondition in the subject. In some embodiments, the administration of theconsecutive dose leads to a further reduction in burden of the diseaseor condition in the subject. In some embodiments, administration of theconsecutive dose leads to a reduction in burden of the disease orcondition in the subject as compared with immediately prior toinitiation of the administration of the consecutive dose. In someembodiments, the method reduces burden of the disease or condition to agreater degree and/or for a greater period of time as compared to amethod with an alternative dosing regimen wherein the subject isadministered the cells in the first dose and the cells in theconsecutive dose in a single dose. The reduction in burden and/orfurther reduction in burden may comprise a reduction in total number ofcells, e.g., tumor cells, of the disease in the subject, in an organ ofthe subject, in a tissue of the subject, or in a bodily fluid of thesubject. The reduction may comprise a reduction in molecular detectionby flow cytometry or quantitative PCR, mass or volume of a tumor, and/ora reduction in number and/or extent of metastases. In some embodiments,the reduction comprises improvement in survival of the subject, e.g.,increased time of survival or incident-free, progression-free, orrelapse-free survival.

In some embodiments, the disease or condition persists following theadministration of the first dose and/or the administration of the firstdose is not sufficient to eradicate the disease or condition in thesubject. In some embodiments, the administration of said consecutivedose leads to a reduction in burden of the disease or condition in thesubject as compared with immediately prior to initiation of theadministration of the consecutive dose.

In some embodiments, the methods reduce burden of the disease orcondition to a greater degree and/or for a greater period of time ascompared to a method comprising an alternative dosing regimen whereinthe subject is administered the cells in (a) (or of the first dose) andthe cells in (b) (or of the consecutive dose) in a single dose. In someembodiments, the area under the curve (AUC) of recombinantreceptor-expressing (e.g., CAR-expressing) cells over time, and/or theduration of detectable receptor-expressing cells in the subjectfollowing the administration of the consecutive dose (or theadministration in (b)) is greater as compared to that achieved via amethod comprising an alternative dosing regimen wherein the subject isadministered the cells in (a) (or the first dose) and the cells in (b)(or the consecutive dose) as a single dose. In some embodiments, themethod results in a maximum concentration or number ofreceptor-expressing (e.g., CAR-expressing) cells in the blood of thesubject of at least at or about 10 receptor-expressing cells permicroliter, at least at or about 100 receptor-expressing cells permicroliter, at least 50% of the total number of peripheral bloodmononuclear cells (PBMCs), at least at least about 1×10⁵receptor-expressing cells, or at least 5,000 copies of recombinantreceptor-encoding (e.g., CAR-encoding) DNA per micrograms DNA, or atleast 10,000 copies of recombinant receptor-encoding DNA per microgramsof DNA. In some embodiments, at day 90 following the initiation of theadministration in (a) or of the first dose, recombinantreceptor-expressing (e.g., CAR-expressing) cells are detectable in theblood or serum of the subject, e.g., the blood of the subject containsat least 10%, at least 20%, at least 40%, or at least 50%, receptor(e.g., CAR)-expressing cells, at least 10 receptor (e.g.,CAR)-expressing cells per microliter or at least 1×10⁴ receptor (e.g.,CAR)-expressing cells.

In some embodiments, the AUC for blood concentration ofreceptor-expressing (e.g., CAR-expressing) cells over time following theadministration in (a) or of the first dose is greater as compared tothat achieved via a method comprising an alternative dosing regimenwherein the subject is administered the cells in (a) or of the firstdose and the cells in (b) or of the second dose as a single dose.

In some embodiments, a CRS-related outcome in the subject at day 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 following the administration in(b) or of the consecutive dose is not detectable or is reduced ascompared to a method comprising an alternative dosing regimen whereinthe subject is administered the cells in (b) or the consecutive dosewithout having been administered the first dose.

In some embodiments, the AUC for a serum level of a factor indicative ofCRS over time in the subject following the administration in (b) or theconsecutive dose is lower as compared to that of a method comprising analternative dosing regimen wherein the subject is administered the cellsin (b) or the consecutive dose without having been administered thefirst dose.

In some embodiments, the subject has been treated with a therapeuticagent targeting the tumor prior to the administration of the first dose.In some aspects, the subject is refractory or non-responsive to saidtherapeutic agent at the time of the administration of the first doseand/or the consecutive dose. In some embodiments, subsequent toadministration in (a) or the first dose and before said administrationin (b) or the consecutive dose, or prior to administration in (a) or thefirst dose, the methods further include assessing a serum level of afactor indicative of CRS, a factor indicative of disease burden, and/oran indicator of a host anti-recombinant receptor (e.g., anti-CAR) immuneresponse in the subject, such as a humoral or cell-mediated immuneresponse. In some such embodiments, the factor indicative of diseaseburden detected is or comprises a total number of cells of the diseasein the subject, in an organ of the subject, in a tissue of the subject,or in a bodily fluid of the subject, molecular detection by flowcytometry or quantitative PCR, mass or volume of a solid tumor, ornumber or extent of metastases.

In some embodiments, the methods include assessing a factor indicativeof disease burden prior to administration of the consecutive dose, andbased on the result of the assessment, determining the consecutive doseof cells to be administered to the subject. In some embodiments, if theassessment determines that the subject has morphologic disease, thesubject is administered a consecutive dose containing less than or aboutthe same number of recombinant receptor- (e.g., CAR-) expressing cellsas the number of recombinant receptor- (e.g., CAR-) expressing cells inthe first dose. In some embodiments, if the assessment determines thatthe subject has minimal residual disease, the subject is administered aconsecutive dose containing an increased number of receptor- (e.g.,CAR-) expressing cells as compared to the first dose. In someembodiments, the consecutive dose comprises about the same number ofrecombinant receptor-expressing (e.g., CAR-expressing) cells as thenumber in the first dose. In some embodiments, the number of receptor-(e.g., CAR-) expressing cells per kilogram administered in theconsecutive dose is less than or about less than or is the same or aboutthe same as the number of receptor- (e.g., CAR-) expressing cells perkilogram administered in the first dose. In other embodiments, thenumber of receptor-expressing (e.g., CAR-expressing) cells per kilogramadministered in the consecutive dose is greater than the number ofreceptor-expressing (e.g., CAR-expressing) cells per kilogramadministered in the first dose. In some embodiments, the consecutivedose comprises an increased number of such cells as compared to thefirst dose, such as at least 2-fold, 5-fold, or 10-fold greater than thenumber in the first dose. In some embodiments, the number ofreceptor-expressing (e.g., CAR-expressing) cells per kilogramadministered in the consecutive dose is at least at or about 2 times orat or about 3 times greater than the number of receptor-expressing(e.g., CAR-expressing) cells per kilogram administered in the firstdose.

In some embodiments, the receptor-expressing (e.g., CAR-expressing)cells in the first dose expand in the subject following administrationof the first dose and/or following the administration of the consecutivedose. In some embodiments, the expansion is evidenced by an increase inserum CRP level following the administration of the first dose and/orconsecutive dose as compared to just prior to the administration. Insome embodiments, the expansion is evidenced by an increase in a levelof receptor- (e.g., CAR-) encoding nucleic acid in the serum, asmeasured by qPCR, following the administration of the first dose and/orconsecutive dose as compared to just prior to the administration. Insome embodiments, the increase is at least 1, 2, or 3-fold.

In some embodiments, the first and/or consecutive dose is not a splitdoes. For example, in some embodiments, the cells of the first dose areadministered in a single pharmaceutical composition comprising the cellsof the first dose and/or the cells of the consecutive dose areadministered in a single pharmaceutical composition comprising the cellsof the consecutive dose. In other embodiments, the first and/orconsecutive dose is a split dose, for example, where the cells of thefirst dose are administered in a plurality of compositions, collectivelycomprising the cells of the first dose, over a period of no more thanthree days; and/or the consecutive dose is a split dose, where the cellsof the consecutive dose are administered in a plurality of compositions,collectively comprising the cells of the consecutive dose, over a periodof no more than three days.

In some embodiments, the methods include administering a consecutivedose of cells expressing a recombinant receptor (e.g., a chimericantigen receptor (CAR)) to a subject previously administered a firstdose of cells expressing a CAR. In some embodiments, the consecutivedose of cells is administered at a time point that is at least or morethan about 14 days after and less than about 28 days after initiation ofthe first dose. In some embodiments, the number of receptor- (e.g.,CAR-) expressing cells administered in the consecutive dose is increasedas compared to the first dose.

In some embodiments, the number of cells administered in the first doseis between about 0.5×10⁶ cells/kg body weight of the subject and 3×10⁶cells/kg, between about 0.75×10⁶ cells/kg and 2.5×10⁶ cells/kg orbetween about 1×10⁶ cells/kg and 2×10⁶ cells/kg, each inclusive.

In some embodiments, the number of cells administered in the consecutivedose of receptor- (e.g., CAR-) expressing cells is between about 2×10⁶cells per kilogram (cells/kg) body weight and about 6×10⁶ cells/kg,between about 2.5×10⁶ cells/kg and about 5.0×10⁶ cells/kg, or betweenabout 3.0×10⁶ cells/kg and about 4.0×10⁶ cells/kg, each inclusive.

In some embodiments, the number of receptor- (e.g., CAR-) expressingcells administered in the first dose is at or about or no more than ator about 1×10⁶ per kilogram of the subject and/or the number ofreceptor- (e.g., CAR-) expressing cells administered in the consecutivedose is at or about 3×10⁶ per kilogram of the subject.

In some embodiments, the methods further include administering achemotherapeutic agent prior to the administration in (a) or prior tothe first dose and/or prior to the administration in (b) or prior to theconsecutive dose. In some embodiments, the subject has been previouslytreated with a chemotherapeutic agent prior to the administration in(a). In some embodiments, the chemotherapeutic agent is or comprises aconditioning chemotherapy, which reduces burden of the disease orcondition in the subject prior to the first dose and/or consecutivedose. In some embodiments, the chemotherapeutic agent iscyclophosphamide, fludarabine, and/or a combination thereof. In someembodiments, the administration of the chemotherapeutic agent includesadministration of a chemotherapeutic agent prior to the administrationof the first dose and optionally not prior to the administration of theconsecutive dose. In some embodiments, the chemotherapeutic agent isadministered between 2 and 5 days prior to the administration of thefirst dose. In some embodiments, the chemotherapeutic agent isadministered between 2 and 5 days prior to the administration of thesecond dose. In some embodiments, the chemotherapeutic agent isadministered between 2 and 5 days prior to the administration first doseand between 2 and 5 days prior to the administration of the second dose.In some embodiments, the chemotherapeutic agent is administered at adose of between at or about 1 g/m² of the subject and at or about 3 g/m²of the subject.

In some embodiments, the subject has received cryoreductive chemotherapyprior to the administration or the first dose. In some embodiments, themethod further includes the administration of cryoreductive chemotherapyprior to the administration of the first dose.

Also provided are cells and compositions for use and uses of cells andcompositions for treating a disease or condition in a subject, such as atumor or cancer, where said cells contain recombinant receptor- (e.g.,chimeric antigen receptor (CAR)-) expressing cells for treatment of adisease or condition in a subject or for the manufacture of a medicamentfor treatment of a disease or condition in a subject previously treatedwith recombinant receptor-expressing (e.g., CAR-expressing) cells. Insome embodiments, the compositions or cells for use or medical uses arefor use 14 to 28 days after the previous treatment. In some embodiments,the compositions or cells for use are formulated for administration of aconsecutive dose in an amount sufficient for reduction in burden of adisease or condition in the subject having been previously treated withthe recombinant receptor-expressing (e.g., CAR-expressing) cells.

In some embodiments of such medical uses, the compositions or cells arefor use that includes administering to a subject having the disease orcondition a first dose of cells expressing the receptor (e.g., CAR). Insome embodiments, the first dose contains no more than about 1×10⁶ ofthe cells per kilogram body weight of the subject, no more than about1×10⁸ of the cells, and/or no more than about 1×10⁸ of the cells/m² ofthe subject. In some embodiments, the compositions or cells are for usethat includes administering to the subject a consecutive dose of cellsexpressing a receptor (e.g., CAR) at a time point that is at least ormore than about 14 days after and less than about 28 days afterinitiation of the administration of the first dose.

In some embodiments, the cells for use are cells expressing arecombinant receptor (e.g., chimeric antigen receptor (CAR)) are for usein methods of treating a disease in a subject previously treated withreceptor- (e.g., CAR-) expressing cells. In some embodiments, the cellsare for use between about 14 and 28 days after the previous treatment.In some embodiments, the cells for use are formulated for administrationof a consecutive dose in an amount sufficient for reduction in burden ofa disease or condition in the subject having been previously treatedwith the receptor- (e.g., CAR-) expressing cells.

In some embodiments of any such compositions or cells for use or medicaluses, the subject does not exhibit morphologic disease and/or thesubject does not exhibit greater than 5% blast cells in the bone marrow.

In some embodiments, the compositions or cells are for use in a methodincluding administering to a subject having the disease or condition afirst dose of cells expressing the receptor (e.g., CAR). In someembodiments, the first dose contains no more than about 1×10⁶ of thecells per kilogram body weight of the subject, no more than about 1×10⁸of the cells, and/or no more than about 1×10⁸ of the cells/m² of thesubject. In some embodiments, the cells are for use in a method thatincludes administering to the subject a consecutive dose of cellsexpressing a receptor (e.g., CAR) at a time point that is at least ormore than about 14 days after and less than about 28 days afterinitiation of said administration of the first dose.

In some embodiments, the compositions or cells for use are formulatedfor administration in an amount that does not induce severe CRS in thesubject or does not induce CRS in the subject. In some embodiments, thecells for use are formulated for administration in an amount that doesnot induce grade 3 or higher neurotoxicity in the subject. In someembodiments, the cells for use are formulated for administration in anamount that, based on clinical data, does not induce severe CRS in amajority of subjects so-treated. In some embodiments, the cells for useare formulated for administration in an amount that, based on clinicaldata, does not induce grade 3 or higher neurotoxicity in a majority ofsubjects so-treated.

In some embodiments, the use of cells expressing a receptor (e.g.,chimeric antigen receptor (CAR)) are for manufacture of a medicament forthe treatment of a disease or condition in a subject includes cells thatare formulated and/or packaged for administration to the subject in afirst and a consecutive dose. In some embodiments, the treatmentincludes administering the cells to the subject in a first and aconsecutive dose, where the first dose includes no more than about 1×10⁶of the cells per kilogram body weight of the subject, no more than about1×10⁸ of the cells, and/or no more than about 1×10⁸ of the cells/m² ofthe subject.

In some embodiments of any such compositions or cells for use or medicaluses, the consecutive dose is for administration at a time point that isat least or more than about 14 days after and less than about 28 daysafter initiation of the first administration. In some embodiments, theconsecutive dose is for administration at a time point at which theserum level in the subject of a factor indicative of cytokine releasesyndrome (CRS) is less than about 10 times, less than about 25 times,and/or less than about 50 times that in the subject immediately prior tosaid first administration. In some embodiments, the consecutive dose isfor administration at a time point at which the subject does not exhibitgrade 3 or higher neurotoxicity. In some embodiments, the consecutivedose is for administration at a time point at which a CRS-relatedoutcome or symptom of neurotoxicity in the subject following saidadministration of said first dose has reached a peak level and begun todecline following the first administration. In some embodiments, theconsecutive dose is for administration at a time point at which thesubject does not exhibit a detectable humoral or cell-mediated immuneresponse against the receptor (e.g., CAR) expressed by the cells of saidfirst dose.

In some embodiments, the cells for use are formulated and/or packagedfor administration to the subject in a first and a consecutive doseand/or the treatment includes administering the cells to the subject ina first and a consecutive dose. In some embodiments, the first dosecontains no more than about 1×10⁶ of the cells per kilogram body weightof the subject, no more than about 1×10⁸ of the cells, and/or no morethan about 1×10⁸ of the cells/m² of the subject.

In some embodiments, the use includes where the first and consecutiveadministrations include administering the cells in one or more unitdose, each unit dose comprising about between 5×10⁷ of the cells andabout 5×10⁸ cells, about between 5×10⁷ of the cells and about 2.5×10⁸cells or about between 2.5×10⁸ cells and 4×10⁸ cells. In someembodiments, the cells are formulated in a unit dose comprising no morethan about 5×10⁷ cell, no more than about 1×10⁸ cells, no more thanabout 2×10⁸ of the cells, no more than about 2.5×10⁸ of the cells, nomore than about 3.0×10⁸ of the cells or no more than about 4×10⁸ of thecells.

In some embodiments, the cells or use includes where the firstadministration comprises administering a single unit dose. In someembodiments, the cells or use includes where the consecutiveadministration comprises administration of two or more unit doses. Insome embodiments, the cells or use includes where the consecutiveadministration comprises administration a single unit dose.

In some embodiments, the cells composition for use or use includes wherethe disease or condition is a tumor or a cancer. In some embodiments,the cells or composition for use or use includes where the tumor orcancer is leukemia or lymphoma. In some embodiments, the composition orcells are for use in treating acute lymphoblastic leukemia. In someembodiments, the composition or cells are for use in treatingnon-Hodgkin lymphoma (NHL).

In some embodiments, the cells, composition, or use includes where theconsecutive dose is formulated for administration of less than or aboutthe same number of recombinant receptor-expressing (e.g.,CAR-expressing) cells as the number of recombinant receptor-expressing(e.g., CAR-expressing) cells in the previous dose. In some embodiments,the composition containing the cells of the consecutive dose isformulated for administration of an increased number of recombinantreceptor-expressing (e.g., CAR-expressing) cells as compared to thefirst dose or previous dose.

Also provided are articles of manufacture for carrying out the methods.In some embodiments, the article of manufacture includes a plurality ofcontainers, e.g., sealable containers, each individually comprising aunit dose of cells expressing the recombinant receptor, e.g., chimericantigen receptor (CAR), for administration to the subject, packagingmaterial, and/or a label or package insert.

In some embodiments, the unit dose comprises the amount of cells to begiven in the lowest dose in the methods, such as the size of the firstdose. In some embodiments, the unit dose includes no more than about1×10⁸ of the cells, no more than about 5×10⁷ of the cells, no more thanabout 1×10⁶ of the cells per kg of the subject, or no more than about5×10⁵ of the cells per kg of the subject.

In some embodiments, the label or package insert includes instructionsfor administering a plurality of the unit doses to the subject, forexample, by administering a certain number of such unit doses, e.g., oneunit dose, in administration of a first dose, and then administering aconsecutive dose including one or a plurality of the unit doses. In someembodiments, the instructions specify carrying out a firstadministration, said first administration comprising delivering one ofsaid unit doses to the subject, and carrying out a consecutiveadministration, said consecutive administration comprising administeringone or a plurality of said unit doses to the subject. In someembodiments, they specify that the consecutive administration is to becarried out at a time between about 15 and about 27 days following saidfirst administration. In some embodiments, the instructions specify thatthe consecutive administration is to be carried out at a time afterwhich it has been determined that a serum level of a factor indicativeof cytokine-release syndrome (CRS) in the subject is less than about 10times, less than about 25 times, and/or less than about 50 times theserum level of said indicator in the subject immediately prior to saidprevious dose, or at a time after which it has been determined that anindicator of CRS has peaked and is declining, such as is at less than ator about 40%, 30%, 20%, or 10% of the peak value, and/or that thesubject does not exhibit a detectable adaptive host immune responsespecific for the receptor (e.g., CAR) expressed by the cells of theprevious dose. In some embodiments, the containers are or compriseflexible cell infusion bags. In some embodiments, the cells to beadministered and/or in the containers or unit doses are for autologoustransfer. Thus, in some embodiments, the cells have been derived fromthe subject to which they are to be administered. In some embodiments,the methods are for allogeneic administration. In some embodiments, thelabel and/or packaging material further includes an identifier specificto the subject, indicating that the cells were derived from the subjectand/or should be administered to the subject specifically.

In some embodiments, the cells are primary cells, such as primary humanimmune cells, e.g., PBMCs, T cells, and/or NK cells. In someembodiments, the cells comprise CD8+ and/or CD4+ T cells. In someembodiments, the T cells are autologous to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows disease responses of subjects with morphological ormolecular disease (disease burden at time of treatment initiation)treated with a single infusion of varying doses of CAR-expressing Tcells. MRD−CR=no minimal residual disease, complete remission;MRD+CR=minimal residual disease, complete remission; NR=not responsive

FIG. 1B shows the presence (Y) or absence (N) of severe cytokine releasesyndrome (CRS) in subjects with morphological or molecular disease(disease burden at time of treatment initiation) treated with a singleinfusion of varying doses of CAR-expressing T cells.

FIG. 1C shows the presence (Y) or absence (N) of severe neurotoxicity insubjects with morphological or molecular disease (disease burden at timeof treatment initiation) treated with a single infusion of varying dosesof CAR-expressing T cells.

FIG. 2 shows peak C reactive protein (CRP) levels in subjects exhibitingmorphological or molecular disease at the time of administration of afirst dose (Infusion=#1) (left-hand panel) and in subjects exhibitingmorphological or molecular disease at the time of administration of aconsecutive dose (Infusion=#2) (right-hand panel) of CAR-expressing Tcells. Numbers and text/abbreviations (e.g., “MRD−CR”, “MRD+CR,” etc.)shown next to individual data points reflect patient numbers and diseaseburden following the indicated infusion. MRD+=minimum residual disease;MRD−=no minimum residual disease; CR=complete remission.

FIG. 3A depicts surface expression, as detected by flow cytometry, ofPD-1, PD-L1, and PD-L2 on a population of T cells gated for positivesurface expression of CD4 and an anti-CD19 chimeric antigen receptor(CAR) (gating strategy shown in top panel), following incubation for 24hours under various conditions (media, K562-tCD19, K562-tROR1,aCD3/aCD28), as described in Example 8.

FIG. 3B depicts surface expression, as detected by flow cytometry, ofPD-1, PD-L1, and PD-L2 on a population of T cells gated for positivesurface expression of CD4 and negative surface expression of ananti-CD19 chimeric antigen receptor (CAR) (gating strategy shown in toppanel), following incubation for 24 hours under various conditions(media, K562-tCD19, K562-tROR1, aCD3/aCD28), as described in Example 8.

FIG. 4A depicts surface expression, as detected by flow cytometry, ofPD-1, PD-L1, and PD-L2 on a population of T cells gated for positivesurface expression of CD8 and an anti-CD19 chimeric antigen receptor(CAR) (gating strategy shown in top panel), following incubation for 24hours under various conditions (media, K562-tCD19, K562-tROR1,aCD3/aCD28), as described in Example 8.

FIG. 4B depicts surface expression, as detected by flow cytometry, ofPD-1, PD-L1, and PD-L2 on a population of T cells gated for positivesurface expression of CD8 and negative surface expression for ananti-CD19 chimeric antigen receptor (CAR) (gating strategy shown in toppanel), following incubation for 24 hours under various conditions(media, K562-tCD19, K562-tROR1, aCD3/aCD28), as described in Example 8.

FIG. 5A shows the degree of neurotoxicity (Grade 0-2, Grade 3, Grade4-5) observed in subjects treated with a single infusion ofCAR-expressing T cells. Data are plotted as tumor burden (percent marrowblasts) vs. number of CAR-expressing T cells (CD8⁺/EGFR⁺, top panel; orCD4⁺/EGFR⁺, bottom panel) per μL of peripheral blood.

FIG. 5B shows the requirement for care in the intensive care unit (ICU)in subjects treated with a single infusion of CAR-expressing T cells.Data are plotted as tumor burden (percent marrow blasts) vs. number ofCD8⁺/EGFR⁺ (top panel) or CD4⁺/EGFR⁺ (bottom panel) cells per μL ofperipheral blood.

FIG. 6A shows the number of CD8+CAR-expressing T cells (CD8⁺/EGFR⁺) perμL of peripheral blood of subjects treated with a single infusion of2×10⁵ or 2×10⁶ CAR-expressing T cells over a 28 day period following theinfusion, as measured by flow cytometry. Prior to the infusion, subjectswere pre-conditioned with 2 g/m² cyclophosphamide with or without 3doses of 100 mg/m² etoposide (No Flu), or were treated with 60 mg/kg (˜2g/m²) cyclophosphamide and 3 to 5 doses of 25 mg/m² fludarabine (Flu).

FIG. 6B shows the number of CD4+CAR-expressing T cells (CD4⁺/EGFR⁺) perμL of peripheral blood of subjects treated with a single infusion of2×10⁵ or 2×10⁶ CAR-expressing T cells over a 28 day period following theinfusion, as measured by flow cytometry. Prior to the infusion, subjectswere pre-conditioned with 2 g//m² cyclophosphamide with or without 3doses of 100 mg/m² etoposide (No Flu), or were treated with 60 mg/kg (˜2g/m²) cyclophosphamide and 3 to 5 doses of 25 mg/m² fludarabine (Flu).

FIG. 6C shows percent disease-free survival curves for subjects treatedwith a single infusion of 2×10⁵ or 2×10⁶ CAR-expressing T cells. Priorto the infusion, subjects were pre-conditioned with 2 g/m²cyclophosphamide with or without 3 doses of 100 mg/m² etoposide (NoFlu), or were treated with 60 mg/kg (˜2 g/m²) cyclophosphamide and 3 to5 doses of 25 mg/m² fludarabine (Flu).

FIG. 7A shows the number (top panel) and percent (bottom panel) ofCD8+CAR-expressing T cells (CD8⁺/EGFR⁺) per μL of peripheral blood ofsubjects treated with a single infusion of 2×10⁷ CAR-expressing T cellsover a 28 day period following the infusion, as measured by flowcytometry. Prior to the infusion, subjects were pre-conditioned with 2-4g/m² cyclophosphamide with or without 3 doses of 100-200 mg/m² etoposide(No Flu), or were treated with 30-60 mg/kg (˜1-2 g/m²) cyclophosphamideand 3 to 5 doses of 25 mg/m² fludarabine (Cy/Flu).

FIG. 7B shows the number (top panel) and percent (bottom panel) of CD4+CAR-expressing T cells (CD4⁺/EGFR⁺) per μL of peripheral blood ofsubjects treated with a single infusion of 2×10⁷ CAR-expressing T cellsover a 28 day period following the infusion, as measured by flowcytometry. Prior to the infusion, subjects were pre-conditioned with 2-4g/m² cyclophosphamide with or without 3 doses of 100-200 mg/m² etoposide(No Flu), or were treated with 30-60 mg/kg (˜1-2 g/m²) cyclophosphamideand 3 to 5 doses of 25 mg/m² fludarabine (Cy/Flu).

DETAILED DESCRIPTION I. Methods of Treatment with Cells ExpressingRecombinant Receptors

Provided are methods, compositions, and articles of manufacture for usein cell therapy, for the treatment of diseases or conditions includingvarious tumors. The methods involve administering engineered cellsexpressing recombinant receptors designed to recognize and/orspecifically bind to molecules associated with the disease or conditionand result in a response, such as an immune response against suchmolecules upon binding to such molecules. The receptors may includechimeric receptors, e.g., chimeric antigen receptors (CARs), and othertransgenic antigen receptors including transgenic T cell receptors(TCRs).

In particular, the methods involve administering one or more consecutivedoses of cells to subjects having received a first dose, and/oradministering the first and one or more consecutive doses. The dosesgenerally are administered in particular amounts and according toparticular timing parameters. In some embodiments, the methods generallyinvolve administering a first dose of cells, thereby reducing diseaseburden, followed by a consecutive dose of cells, administered during aparticular time window with respect to the first dose, or theadministration of the consecutive dose to a subject having received sucha first dose. The first dose is often a relatively low dose. In someembodiments, additional consecutive doses then are administered, forexample, within the same or a similar window of time with respect to theconsecutive dose. In some embodiments, the number of cells administeredand timing of the multiple doses are designed to improve one or moreoutcomes, such as to reduce the likelihood or degree of toxicity to thesubject, improve exposure of the subject to and/or persistence of theadministered cells, and/or improve therapeutic efficacy. Also providedare articles of manufacture containing the cells and designed foradministration following such dosing regimens.

In some embodiments, the provided methods are based on observationsherein that increased exposure of the subject to the administered cells(e.g., increased number of cells or duration over time) can improveefficacy and therapeutic outcomes in adoptive cell therapy. Preliminaryanalysis conducted following the administration of differentCD19-targeting CAR-expressing T cells to subjects with variousCD19-expressing cancers in multiple clinical trials revealed acorrelation between greater and/or longer degree of exposure to theCAR-expressing cells and treatment outcomes. Such outcomes includedpatient survival and remission, even in individuals with severe tumorburden.

Yet delivering high initial doses of the recombinant immunostimulatorycells does not necessarily increase exposure. Particularly in thecontext of high disease burden (and thus higher amounts of antigen),administering large doses does not necessarily enhance efficacy and canlead to increased or rapid expansion of the cells and result intoxicity. Certain reports have indicated a lack of correlation betweendose and toxicity. See Park et al, Molecular Therapy 15(4):825-833(2007). On the other hand, higher initial doses can promote toxicoutcomes such as cytokine release syndrome (CRS), particularly in thecontext of high disease burden. Moreover, high doses do not necessarilytranslate to increased persistence of the administered cells, and thusdo not necessarily increase exposure over time. See Park et al,Molecular Therapy 15(4):825-833 (2007). Likewise, administering cells inthe context of high disease burden, often present at the outset oftreatment, can lead to exhaustion of the transferred cells, therebyreducing clinical efficacy. See Davila & Brentjens, Hematol Oncol ClinNorth Am. 27(2):341-53 (2013).

Administering subsequent doses may not be effective, particularlyfollowing relapse and/or where the subject has mounted an immuneresponse specific for the cells or the recombinant receptors theexpress. Improved methods are needed to increase cell exposure over timewhile avoiding toxic outcomes.

The provided methods offer advantages over other approaches aimed ataddressing the risk of toxic outcomes and/or improving efficacy. Manysuch approaches have focused, for example, on targeting downstreameffects of toxicity, such as by cytokine blockade, and/or deliveringagents such as high-dose steroids which can also eliminate or impair thefunction of administered cells. Many of these other approaches also donot prevent other forms of toxicity such as neurotoxicity, which can beassociated with adoptive cell therapy. On the other hand, administeringrelatively low doses of cells (e.g. CAR-expressing cells) may decreasethe risk but may not be completely effective. Delivery of subsequentdoses of cells, for example, after relapse following an initialadministration, also has not been entirely satisfactory. Such dosingapproaches can lead to limited or ineffective responses of thesubsequent dose, due to host immune responses mounted against theinitial dose.

Provided are methods involving the administration of consecutive dosesof cell therapy in a way that minimizes risk of toxicity and maximizesefficacy. As observed herein, following administration of differenttypes of CD19-targeting CAR T cells to human subjects with B cellcancers, initial doses equal to or below 1×10⁶ cells per kilogram bodyweight were effective in reducing tumor burden. These low doses alsowere associated with less frequent severe CRS, neurotoxicity, and otheradverse events compared with higher doses. In some embodiments, theprovided methods involve the safe initial administration of such lowdoses, followed by consecutive dose(s) for increased exposure, with doseamounts and timing designed to avoid impairment of efficacy by hostimmune responses while minimizing risk of toxicity

In some cases, if desired or need in a particular disease or context,the provided methods involve a consecutive dose of cells administered atan increased number, and hence at a higher dose, than the first dose ofcells. As shown herein, in some aspects, administration of higher dosesof cells is advantageous compared to lower doses. In some embodiments,administration of higher doses of cells, such as doses greater than orequal to 2.5×10⁶ cells/kg or higher, are associated with an increasedoverall survival in subjects, particularly in subjects that exhibitmorphological disease prior to treatment. In some embodiments, however,these higher doses can be associated with toxic outcomes, and inparticular with neurotoxicity, such as severe neurotoxicity, for examplegrade 3 neurotoxicity or higher, particularly in subjects withmorphological disease prior to treatment. In some cases, the toxicoutcome following administration of high doses of recombinantreceptor-expressing (e.g., CAR-expressing) cells is not observed, or isnot observed to as great of an extent, when subjects with a reduceddisease burden are treated, such as subjects with minimal residualdisease having molecularly detectable disease. In some embodiments,methods of first administering a low dose of recombinantreceptor-expressing (e.g., CAR-expressing) cells can reduce the diseaseburden in subjects, such as from morphological disease status to minimalresidual disease, so that subsequent administration of a higher dose ofrecombinant receptor-expressing (e.g., CAR-expressing) cells in subjectsis less likely to cause toxic outcomes in a majority of subjectstreated. In some embodiments, the provided methods avoid the problems oftoxicity associated with administration with higher doses, whilemaximizing the efficacy of treatment that can occur upon administrationwith higher numbers of recombinant receptor-expressing (e.g.,CAR-expressing) cells.

In some embodiments, the methods include administering an initial doseof recombinant receptor-expressing (e.g., CAR-expressing) cells that canexpand in the presence of disease-associated antigens and reduce diseaseburden but without the same degree of toxic outcomes that may beassociated with a higher dose. In some aspects, the first dose is a lowdose, such as a dose of less than at or about or no more than at orabout 1×10⁶ cells per kilogram body weight of the subject (cells/kg),0.5×10⁶ cells/kg or 1×10⁵ cells/kg. In some aspects, the first dose isin an amount or number of cells that has been observed not to causetoxic outcomes in the disease or condition or subject, such as, in someembodiments, CRS or neurotoxicity. In some embodiments, the first doseis an amount or number of cells that has been associated with suchoutcomes, such as based on clinical data, in only a relatively smallpercentage of patients, such as no more than 50, 40, 25, 20, 15, 10, 5,or fewer percent of subjects.

In some embodiments, the first dose is generally also large enough to beeffective in reducing disease burden. In some cases, the first dose islarge enough to reduce disease burden if the cell dose is sufficient toexpand in vivo and debulk disease. In some embodiments, the cells of thefirst dose thereby debulk or reduce disease burden, e.g., tumor size,without effecting severe unwanted outcomes. In some cases, the firstdose is an amount of cells that is effective to reduce tumor burden,such as by reducing disease from a morphological setting to minimumresidual disease and/or clinical or complete remission. In some aspects,the initial dose is a low dose. In some aspects, for example, in thecontext of relatively low disease burden, the first dose may be higher.

In some aspects, a risk-adapted dosing regime is used for determiningthe appropriate number or amount or relative number or amount of cellsor recombinant receptor-expressing (e.g., CAR-expressing) cells in thedose. For example, in some aspects, prior to infusion of recombinantreceptor-expressing (e.g., CAR-expressing) cells, the disease burden ofthe subject is determined and, based on the disease burden, a first doseof recombinant receptor-expressing (e.g., CAR-expressing) cells (e.g.low or high dose) is selected that can minimize toxicity and maximizeefficacy, for example, based on observations described above andelsewhere herein in which higher doses of recombinantreceptor-expressing cells (e.g., CAR-expressing, such as CAR-expressingT cells) can be associated with toxic outcomes, such as severeneurotoxicity, in subjects having morphological disease burden, whichare not necessarily observed, or are not observed to as great of anextent, in subjects with relatively lower disease burden. For example,subjects with a high marrow tumor burden prior to treatment, which, insome cases, can be associated with a greater CAR T-cell expansionfollowing treatment, were observed to have a higher risk of developingsevere neurotoxicity that may require ICU care.

In some embodiments, in order to maximize efficacy in subjects that arenot, or are less, susceptible to a toxic outcome following in infusionof recombinant receptor-expressing (e.g., CAR-expressing) cells, e.g.CAR-T cell infusion, (such as to subjects that do not exhibitmorphologic disease (i.e. have non-morphologic disease) or do notexhibit substantial morphologic disease), a relatively higher dose ofcells, such as greater than 1×10⁶ cells/kg, 2×10⁶ cells/kg, 5×10⁶cells/kg or 1×10⁷ cells/kg, can be administered. Conversely, in someaspects, subjects that are determined to have a relatively higherdisease burden, such as by the presence of morphologic disease orsubstantial morphologic disease, can be administered a relatively lowerdose of cells than administered to subjects that do not exhibitmorphologic disease or do not exhibit substantial morphologic disease,such as a dose of less than or equal to or about 1×10⁶ cell/kg or lessthan or equal to or about 0.5×10⁶ cells/kg. In some embodiments, one ormore further consecutive doses can be administered, which, optionally,can be administered based on the extent or degree of disease burden asdescribed above or alternatively be at a fixed dose regardless ofdisease burden. In some embodiments, a subject exhibits morphologicdisease or substantial morphological disease burden if there are greaterthan or equal to or about 5% blasts present in the bone marrow. In someembodiments, a subject with higher relative disease burden, such hashigh marrow tumor burden, has greater than, equal to or greater thanabout 10% blasts in the bone marrow or greater than, equal to or greaterthan about 20% blasts in the bone marrow.

In some embodiments, a subject is assessed for disease burden prior totreatment, and, if the subject exhibits less than 5% bone marrow blastcells, the subject can be administered a dose of recombinantreceptor-expressing (e.g., CAR-expressing, such as CAR-expressing Tcells) of greater than 1×10⁶ cells/kg, 2×10⁶ cells/kg, 5×10⁶ cells/kg or1×10⁷ cells/kg, or if the subject exhibits greater than or equal to 5%bone marrow blast cells, the subject can be administered a dose ofrecombinant receptor-expressing (e.g., CAR-expressing, such asCAR-expressing T cells) of less than or equal to or about 1×106 cell/kgor 0.5×106 cells/kg. In some embodiments, a subject is assessed fordisease burden prior to treatment, and, if the subject exhibits lessthan 10% bone marrow blast cells, the subject can be administered a doseof recombinant receptor-expressing (e.g., CAR-expressing, such asCAR-expressing T cells) of greater than 1×10⁶ cells/kg, 2×10⁶ cells/kg,5×10⁶ cells/kg or 1×10⁷ cells/kg, or if the subject exhibits greaterthan or equal to 10% bone marrow blast cells, the subject can beadministered a dose of recombinant receptor-expressing (e.g.,CAR-expressing, such as CAR-expressing T cells) of less than or equal toor about 1×10⁶ cell/kg or 0.5×10⁶ cells/kg. In some embodiments, asubject is assessed for disease burden prior to treatment, and, if thesubject exhibits less than 20% bone marrow blast cells, the subject canbe administered a dose of recombinant receptor-expressing (e.g.,CAR-expressing, such as CAR-expressing T cells) of greater than 1×10⁶cells/kg, 2×10⁶ cells/kg, 5×10⁶ cells/kg or 1×10⁷ cells/kg, or if thesubject exhibits greater than or equal to 20% bone marrow blast cells,the subject can be administered a dose of recombinantreceptor-expressing (e.g., CAR-expressing, such as CAR-expressing Tcells) of less than or equal to or about 1×10⁶ cell/kg or 0.5×10⁶cells/kg. In some embodiments, the disease burden is assessed and arisk-adapted dose is selected prior to the first dose of recombinantreceptor-expressing (e.g., CAR-expressing, such as CAR-expressing Tcells). In some embodiments, the disease burden is assessed and arisk-adapted dose is selected prior to one or more consecutive doses ofrecombinant receptor-expressing (e.g., CAR-expressing, such asCAR-expressing T cells).

In some embodiments, a consecutive dose of recombinantreceptor-expressing (e.g., CAR-expressing) cells is administered to thesubject at a time after administration of the first or initial dose ofcells in which it is likely that tumor burden of the subject has beenreduced by the first dose. In some embodiments, it is not necessary thatthe tumor burden actually be reduced in all subjects prior toadministration of the consecutive dose, but that tumor burden is reducedon average in subjects treated, such as based on clinical data, in whicha majority of subjects treated with such a first dose exhibit a reducedtumor burden, such as at least 50%, 60%, 70%, 80%, 90%, 95% or more ofsubjects treated with the first or initial dose exhibit a reduced tumorburden. Generally at a point in time after disease burden has beenreduced by the first dose or is likely to have been reduced by the firstdose, a consecutive dose is administered to the subject, thereby furtherreducing and/or eliminating disease or a symptom or outcome thereof orpreventing expansion or progression thereof. The context of reduceddisease burden at the time of the consecutive administration in someaspects reduces the likelihood of exhaustion of the transferred cells,thereby improving efficacy. The consecutive dose may be the same, lower,or a higher dose as compared with the first dose. In some embodiments,multiple consecutive doses are administered after a first dose.

In some embodiments, the consecutive dose of recombinantreceptor-expressing (e.g., CAR-expressing) cells is administered at adose that is higher than the first dose so that an increased number ofrecombinant receptor-expressing (e.g., CAR-expressing) cells isadministered to the subject by the consecutive dose. In someembodiments, a higher dose of recombinant receptor-expressing (e.g.,CAR-expressing) cells is one that can promote an increased response orefficacy, such as improved or greater reduction in tumor burden and/oran improved or greater overall survival time of the subject compared tothat achieved by administration of a lower dose or number of cells. Insome embodiments, because administration of the first dose of cells canreduce tumor burden in the subject, administration of the consecutivedose at a higher number of cells can avoid or minimize CRS and/orneurotoxicity in the subject after administration of the consecutivedose that can otherwise occur in subjects with morphological disease.

In some embodiments, prior to administration of the consecutive dose,the subject is one that exhibits non-morphological disease, such asmolecularly detectable disease and/or minimal residual disease. In someembodiments, a subject can be assessed for tumor burden afteradministration of the first dose and prior to administration of theconsecutive dose to confirm that tumor burden has been reduced comparedto tumor burden present prior to treatment with the first dose. In someembodiments, if assessment of the subject indicates tumor burden isreduced and/or that the subject exhibits non-morphological disease, forexample molecularly detectable disease and/or minimal residual disease,the provided methods include administering a consecutive dose ofrecombinant receptor-expressing (e.g., CAR-expressing) cells that ishigher than the first or initial dose. In some embodiments, ifassessment of the subject indicates tumor burden is not reduced and/orthe subject exhibits morphological disease, the provided methods includeadministering a consecutive dose of recombinant receptor-expressing(e.g., CAR-expressing) cells that is the same as or less than the firstor initial dose.

In some embodiments, the timing of the consecutive dose(s) in relationto the first and/or one another is designed to reduce the risk ofunwanted toxic outcomes and promote maximum efficacy. In someembodiments, the consecutive dose, such as the same, lower or higherconsecutive dose, is administered at a time at which disease burdenremains reduced in the subject or reduced in subjects on average, suchas based on clinical data, but at which the risk of CRS and/orneurotoxicity remain low. In some embodiments, methods are provided inwhich CRS and/or neurotoxicity in a subject can be prevented, minimizedor reduced by first administering a low dose of CAR+ T cells to reducedisease or tumor burden to non-morphological disease, for example toachieve minimal residual disease or molecular detectable disease status,followed by administration of the consecutive dose of recombinantreceptor-expressing (e.g., CAR-expressing) cells.

In some embodiments, a consecutive dose is generally given at a point intime relative to the first or previous dose at which the risk of a toxicoutcome or symptom or biochemical indicator thereof—such as CRS orneurotoxicity, macrophage activation syndrome, or tumor lysissyndrome—is at or below an acceptable level. For example, theconsecutive dose may be administered after a toxic outcome has peakedand is declining or has declined below an acceptable level following theinitial dose. Thus, in some embodiments, the consecutive dose isadministered at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 days following the initiationof the first or prior dose, or greater than about 14 or 15 days or 21days following the initiation of the first or prior dose. In someembodiments, the appropriate timing is determined by monitoring and/orassessing the presence of one or more symptoms or outcomes associatedwith the toxic event and delivering the consecutive dose afterdetermining that the symptom or outcome is at or below an acceptablelevel.

In some embodiments, the timing of the consecutive dose is such that itavoids a reduction in efficacy that may otherwise be induced uponadministration of a subsequent dose by a host immune response that hasbeen mounted following administration of a first dose. In some aspects,the consecutive dose is administered prior to the development of a hostimmune response, e.g., adaptive or specific, e.g., humoral orcell-mediated, immune response against the administered cells and/orrecombinant receptor they express, and/or before such a response isdetectable, e.g., by one or more specified detection methods. Theconsecutive dose or doses generally are administered at a time at whicha host adaptive immune response against the cells is not detected, hasnot become established, and/or has not reached a certain level or degreeor stage. In this regard, the methods are advantageous compared toproviding subsequent doses at the time of relapse, which generally isafter an anti-transgene response has developed. In some embodiments, theconsecutive dose is administered before or within about 28 days or 35days following the first or prior dose, or before about 24, 25, 26, or27 days following the initiation of the first or prior dose.

Thus, the provided methods in some embodiments involve administering oneor more consecutive doses after debulking or reducing disease burdenwith a first dose, after a window of risk for toxicity, but before themounting of an immune response. In this environment and with theappropriate timing, the consecutive dose can safely and effectivelyprovide immune surveillance, clearing or preventing expansion ormetastasis of residual disease cells, whether measurable or not bystandard or research grade analytical methods. Thus, in someembodiments, the consecutive dose is a disease-consolidating dose.

In particular embodiments, the first dose includes the cells in anamount sufficient to reduce burden of the disease or condition in thesubject and the consecutive dose is administered a time at which a serumlevel of a factor indicative of cytokine-release syndrome (CRS) in thesubject is no more than 10 or no more than 25 times the serum level ofthe indicator in the subject immediately prior to the administration ofthe first dose, and/or at a time after a CRS-related outcome in thesubject has reached a peak level and begun to decline followingadministration of the first dose, and at which the subject does notexhibit a detectable adaptive host immune response specific for therecombinant receptor expressed by the cells of the first dose.

In particular embodiments, the first dose contains fewer than at orabout 1×10⁶ of the cells per kilogram body weight of the subject, fewerthan at or about 1×10⁸ of the cells, and/or fewer than at or about 1×10⁸of the cells/m² of the subject, and the consecutive dose is administeredat a time point that is more than about 14 days after and less thanabout 28 days after initiation of the administration of the first dose.In some embodiments, the consecutive dose is administered at or about 14days, 15 days, 16, days, 17 days, 18 days, 19 days, 20 days, 21 days, 22days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days followingthe first or prior dose. In particular embodiments, the consecutive doseis administered at about 17 days or at about 21 days following the firstor prior dose.

Thus, the provided methods offer advantages over single-doseadministrations, which can lead to severe toxicity, unwanted outcomes,and/or lower efficacy, particularly in the context of high diseaseburden. They also can be advantageous over methods administeringsubsequent dose(s) too soon following an initial dose—increasing therisk of unwanted side effects—or too late, e.g., after establishment ofan immune response to a previous dose. The provided methods extendexposure to therapeutic cells, improving durability and extent ofclinical response and/or patient survival, while reducing toxicoutcomes. Also provided are compositions and articles of manufactureproviding doses for use in such methods.

II. Administration of Cells in Adoptive Cell Therapy

The provided methods generally involve administering multiple doses ofcells expressing recombinant receptors, such as CARs, or other antigenreceptors, such as transgenic TCRs, to subjects having a disease orcondition specifically recognized by the receptors. The administrationsgenerally effect an improvement in one or more symptoms of the diseaseor condition and/or treat or prevent the disease or condition or symptomthereof.

As used herein, a “subject” is a mammal, such as a human or otheranimal, and typically is human. In some embodiments, the subject hasbeen treated with a therapeutic agent targeting the disease orcondition, e.g. the tumor, prior to administration of the first doseand/or prior to the administration of the consecutive dose. In someaspects, the subject is refractory or non-responsive to the othertherapeutic agent.

In some embodiments, the subject has persistent or relapsed disease,e.g., following treatment with another therapeutic intervention,including chemotherapy, radiation, and/or hematopoietic stem celltransplantation (HSCT), e.g., allogenic HSCT. In some embodiments, theadministration effectively treats the subject despite the subject havingbecome resistant to another therapy.

In some embodiments, the subject is responsive to the other therapeuticagent, and treatment with the therapeutic agent reduces disease burden.In some aspects, the subject is initially responsive to the therapeuticagent, but exhibits a relapse of the disease or condition over time. Insome embodiments, the subject has not relapsed. In some suchembodiments, the subject is determined to be at risk for relapse, suchas at a high risk of relapse, and thus the cells are administeredprophylactically, e.g., to reduce the likelihood of or prevent relapse.

In some aspects, the subject has not received prior treatment withanother therapeutic agent. In some embodiments, the subject has notreceived a dose of cells expressing a CAR prior to the administration ofthe first dose and/or has not received a dose of cells expressing theCAR or other receptor expressed by such cells or expressing anyrecombinant receptor targeting the same molecule or antigen. In someaspects, the subject has not received a dose of cells expressing the CARof the first dose prior to the administration of the first dose.

Among the diseases, conditions, and disorders are tumors, includingsolid tumors, hematologic malignancies, and melanomas, and includinglocalized and metastatic tumors, infectious diseases, such as infectionwith a virus or other pathogen, e.g., HIV, HCV, HBV, CMV, and parasiticdisease, and autoimmune and inflammatory diseases. In some embodiments,the disease or condition is a tumor, cancer, malignancy, neoplasm, orother proliferative disease or disorder. Such diseases include but arenot limited to leukemia, lymphoma, e.g., chronic lymphocytic leukemia(CLL), acute-lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, acutemyeloid leukemia, multiple myeloma, refractory follicular lymphoma,mantle cell lymphoma, indolent B cell lymphoma, B cell malignancies,cancers of the colon, lung, liver, breast, prostate, ovarian, skin,melanoma, bone, and brain cancer, ovarian cancer, epithelial cancers,renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma,cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma,Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and/ormesothelioma. In some embodiments, the subject has acute-lymphoblasticleukemia (ALL). In some embodiments, the subject has non-Hodgkin'slymphoma.

In some embodiments, the size or timing of the doses is determined as afunction of the particular disease or condition in the subject. It iswithin the level of a skilled artisan to empirically determine the sizeor timing of the doses for a particular disease in view of the provideddescription. In some cases, for example, subjects with non-Hodgkin'slymphoma (NHL) can be less responsive or only partially responsive totreatment with recombinant receptor-expressing cells (e.g.,CAR-expressing cells, such as CAR-expressing T cells) than subjects withacute-lymphoblastic leukemia (ALL). In some cases, a lower dose of cellsadministered to a subject with NHL may not reduce tumor burden in asubject, thereby increasing the risk of toxic outcomes, such as CRSand/or neurotoxicity, in a subject when administered a consecutive dose.In some embodiments, methods are provided in which subjects with NHL areadministered a consecutive dose that is the same or less than the doseof recombinant receptor-expressing (e.g., CAR-expressing) cellsadministered in the first dose, thereby minimizing or reducing risk oftoxic side effects that can occur upon administration of the consecutivedose.

In some embodiments, the disease or condition is a tumor or a cancer andthe subject has a large tumor burden prior to the administration of thefirst dose, such as a large solid tumor or a large number or bulk ofdisease-associated, e.g., tumor or cancer cells. In some aspects, thesubject has a high number of metastases and/or widespread localizationof metastases. In some aspects, the initial tumor burden in the subjectis low and the subject has few metastases.

In some embodiments, the size or timing of the doses is determined bythe initial disease burden, such as tumor burden, in the subject. Forexample, in some cases, whereas the subject generally is administered arelatively low number of cells in the first dose, in context of lowerdisease burden, such as molecular detectable disease and/or minimalresidual disease, the initial dose may be higher. In other cases, insubjects exhibiting a higher disease burden after administration of thefirst dose, the consecutive dose may be the same as or lower than thefirst dose. In some embodiments, subjects are assessed for diseaseburden using methods as described herein, such as methods that assessblasts in bone marrow or molecular disease by flow cytometry or qPCRmethods.

In some embodiments, the disease or condition is an infectious diseaseor condition, such as, but not limited to, viral, retroviral, bacterial,and protozoal infections, immunodeficiency, Cytomegalovirus (CMV),Epstein-Ban virus (EBV), adenovirus, BK polyomavirus. In someembodiments, the disease or condition is an autoimmune or inflammatorydisease or condition, such as arthritis, e.g., rheumatoid arthritis(RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatorybowel disease, psoriasis, scleroderma, autoimmune thyroid disease,Grave's disease, Crohn's disease, multiple sclerosis, asthma, and/or adisease or condition associated with transplant.

In some embodiments, the antigen associated with the disease or disorderis selected from the group consisting of orphan tyrosine kinase receptorROR1, tEGFR, Her2, Ll-CAM, CD19, CD20, CD22, mesothelin, CEA, andhepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30,CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP, fetalacethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha,IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesionmolecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands,NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2,carcinoembryonic antigen (CEA), prostate specific antigen, PSMA,Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123,CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin,such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/ormolecules expressed by HIV, HCV, HBV or other pathogens.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to complete or partial amelioration orreduction of a disease or condition or disorder, or a symptom, adverseeffect or outcome, or phenotype associated therewith. Desirable effectsof treatment include, but are not limited to, preventing occurrence orrecurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis.The terms do not imply complete curing of a disease or completeelimination of any symptom or effect(s) on all symptoms or outcomes.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, suppress and/or postpone development ofthe disease (such as cancer). This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop the disease. For example, a late stagecancer, such as development of metastasis, may be delayed.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease. In some embodiments, the provided cells and compositionsare used to delay development of a disease or to slow the progression ofa disease.

As used herein, to “suppress” a function or activity is to reduce thefunction or activity when compared to otherwise same conditions exceptfor a condition or parameter of interest, or alternatively, as comparedto another condition. For example, cells that suppress tumor growthreduce the rate of growth of the tumor compared to the rate of growth ofthe tumor in the absence of the cells.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,cells, or composition, in the context of administration, refers to anamount effective, at dosages/amounts and for periods of time necessary,to achieve a desired result, such as a therapeutic or prophylacticresult.

A “therapeutically effective amount” of an agent, e.g., a pharmaceuticalformulation or cells, refers to an amount effective, at dosages and forperiods of time necessary, to achieve a desired therapeutic result, suchas for treatment of a disease, condition, or disorder, and/orpharmacokinetic or pharmacodynamic effect of the treatment. Thetherapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the subject, and thepopulations of cells administered. In some embodiments, the providedmethods involve administering the cells and/or compositions at effectiveamounts, e.g., therapeutically effective amounts.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount. In the context of lower tumor burden, theprophylactically effective amount in some aspects will be higher thanthe therapeutically effective amount.

Methods for administration of cells for adoptive cell therapy are knownand may be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

In some embodiments, the cell therapy, e.g., adoptive cell therapy,e.g., adoptive T cell therapy, is carried out by autologous transfer, inwhich the cells are isolated and/or otherwise prepared from the subjectwho is to receive the cell therapy, or from a sample derived from such asubject. Thus, in some aspects, the cells are derived from a subject,e.g., patient, in need of a treatment and the cells, and followingisolation and processing are administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive cell therapy,e.g., adoptive T cell therapy, is carried out by allogeneic transfer, inwhich the cells are isolated and/or otherwise prepared from a subjectother than a subject who is to receive or who ultimately receives thecell therapy, e.g., a first subject. In such embodiments, the cells thenare administered to a different subject, e.g., a second subject, of thesame species. In some embodiments, the first and second subjects aregenetically identical. In some embodiments, the first and secondsubjects are genetically similar. In some embodiments, the secondsubject expresses the same HLA class or supertype as the first subject.

The cells can be administered by any suitable means, for example, bybolus infusion, by injection, e.g., intravenous or subcutaneousinjections, intraocular injection, periocular injection, subretinalinjection, intravitreal injection, trans-septal injection, subscleralinjection, intrachoroidal injection, intracameral injection,subconjectval injection, subconjuntival injection, sub-Tenon'sinjection, retrobulbar injection, peribulbar injection, or posteriorjuxtascleral delivery. In some embodiments, they are administered byparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In some embodiments, a given dose isadministered by a single bolus administration of the cells. In someembodiments, it is administered by multiple bolus administrations of thecells, for example, over a period of no more than 3 days, or bycontinuous infusion administration of the cells.

For the prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the type of cells orrecombinant receptors, the severity and course of the disease, whetherthe cells are administered for preventive or therapeutic purposes,previous therapy, the subject's clinical history and response to thecells, and the discretion of the attending physician. The compositionsand cells are in some embodiments suitably administered to the subjectat one time or over a series of treatments.

In some embodiments, the cells are administered as part of a combinationtreatment, such as simultaneously with or sequentially with, in anyorder, another therapeutic intervention, such as an antibody orengineered cell or receptor or agent, such as a cytotoxic or therapeuticagent. The cells in some embodiments are co-administered with one ormore additional therapeutic agents or in connection with anothertherapeutic intervention, either simultaneously or sequentially in anyorder. In some contexts, the cells are co-administered with anothertherapy sufficiently close in time such that the cell populationsenhance the effect of one or more additional therapeutic agents, or viceversa. In some embodiments, the cells are administered prior to the oneor more additional therapeutic agents. In some embodiments, the cellsare administered after the one or more additional therapeutic agents. Insome embodiments, the one or more additional agent includes a cytokine,such as IL-2, for example, to enhance persistence.

In some embodiments, the methods comprise administration of achemotherapeutic agent, e.g., a conditioning chemotherapeutic agent, forexample, to reduce tumor burden prior to the first or consecutive doseadministrations.

Preconditioning subjects with immunodepleting (e.g., lymphodepleting)therapies can improve the effects of adoptive cell therapy (ACT).Preconditioning with lymphodepleting agents, including combinations ofcyclosporine and fludarabine, have been effective in improving theefficacy of transferred tumor infiltrating lymphocyte (TIL) cells incell therapy, including to improve response and/or persistence of thetransferred cells. See, e.g., Dudley et al., 2002 Science, 298, 850-54;Rosenberg et al., Clin Cancer Res 2011, 17(13):4550-4557. Likewise, inthe context of CAR+ T cells, several studies have incorporatedlymphodepleting agents, most commonly cyclophosphamide, fludarabine,bendamustine, or combinations thereof, sometimes accompanied by low-doseirradiation. See Han et al. Journal of Hematology & Oncology 2013, 6:47;Kochenderfer et al., Blood 2012; 119: 2709-2720; Kalos et al., SciTransl Med 2011, 3(95):95ra73; Clinical Trial Study Record Nos.:NCT02315612; NCT01822652. Such preconditioning can be carried out withthe goal of reducing the risk of one or more of various outcomes thatcould dampen efficacy of the therapy. These include the phenomenon knownas “cytokine sink,” by which T cells, B cells, NK cells compete withTILs for homeostatic and activating cytokines, such as IL-2, IL-7,and/or IL-15; suppression of TILs by regulatory T cells, NK cells, orother cells of the immune system; impact of negative regulators in thetumor microenvironment. Muranski et al., Nat Clin Pract Oncol. 2006December; 3(12): 668-681.

Thus, in some embodiments, the methods include administering apreconditioning agent, such as a lymphodepleting or chemotherapeuticagent, such as cyclophosphamide, fludarabine, or combinations thereof,to a subject prior to the first or subsequent dose. For example, thesubject may be administered a preconditioning agent at least 2 daysprior, such as at least 3, 4, 5, 6, or 7 days prior, to the first orsubsequent dose. In some embodiments, the subject is administered apreconditioning agent no more than 7 days prior, such as no more than 6,5, 4, 3, or 2 days prior, to the first or subsequent dose

In some embodiments, where the lymphodepleting agent comprisescyclophosphamide, the subject is administered between or between about0.5 g/m² and 5 g/m², such as between or between about 1 g/m² and 4 g/m²,1 g/m² and 3 g/m², or 2 g/m² and 4 g/m² of cyclophosphamide. In someaspects, the subject is administered 2 g/m² cyclophosphamide or about 2g/m² cyclophosphamide. In some embodiments, the subject ispreconditioned with cyclophosphamide at a dose between or between about20 mg/kg and 100 mg/kg, such as between or between about 40 mg/kg and 80mg/kg. In some aspects, the subject is preconditioned with or with about60 mg/kg of cyclophosphamide.

In some embodiments, where the lymphodepleting agent comprisesfludarabine, the subject is administered fludarabine at a dose betweenor between about 1 g/m² and 100 g/m², such as between or between about10 g/m² and 75 g/m², 15 g/m² and 50 g/m², 20 g/m² and 30 g/m², or 24g/m² and 26 g/m². In some instances, the subject is administered 25 g/m²of fludarabine. In some embodiments, the fludarabine can be administeredin a single dose or can be administered in a plurality of doses, such asgiven daily, every other day or every three days. For example, in someinstances, the agent, e.g., fludarabine, is administered between orbetween about 1 and 5 times, such as between or between about 3 and 5times. In some embodiments, such plurality of doses is administered inthe same day, such as 1 to 5 times or 3 to 5 times daily.

In some embodiments, the lymphodepleting agent comprises a combinationof agents, such as a combination of cyclophosphamide and fludarabine.Thus, the combination of agents may include cyclophosphamide at any doseor administration schedule, such as those described above, andfludarabine at any dose or administration schedule, such as thosedescribed above. For example, in some aspects, the subject isadministered 60 mg/kg (˜2 g/m²) of cyclophosphamide and 3 to 5 doses of25 mg/m² fludarabine prior to the first or subsequent dose.

In some embodiments, the administration of the preconditioning agentprior to infusion of the first or subsequent dose improves an outcome ofthe treatment. For example, in some aspects, preconditioning improvesthe efficacy of treatment with the first or subsequent dose or increasesthe persistence of the recombinant receptor-expressing cells (e.g.,CAR-expressing cells, such as CAR-expressing T cells) in the subject. Insome embodiments, preconditioning treatment increases disease-freesurvival, such as the percent of subjects that are alive and exhibit nominimal residual or molecularly detectable disease after a given periodof time following the first or subsequent dose. In some embodiments, thetime to median disease-free survival is increased.

Once the cells are administered to the subject (e.g., human), thebiological activity of the engineered cell populations in some aspectsis measured by any of a number of known methods. Parameters to assessinclude specific binding of an engineered or natural T cell or otherimmune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., byELISA or flow cytometry. In certain embodiments, the ability of theengineered cells to destroy target cells can be measured using anysuitable method known in the art, such as cytotoxicity assays describedin, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702(2009), and Herman et al. J. Immunological Methods, 285(1): 25-40(2004). In certain embodiments, the biological activity of the cellsalso can be measured by assaying expression and/or secretion of certaincytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects thebiological activity is measured by assessing clinical outcome, such asreduction in tumor burden or load. In some aspects, toxic outcomes,persistence and/or expansion of the cells, and/or presence or absence ofa host immune response, are assessed.

In certain embodiments, engineered cells are modified in any number ofways, such that their therapeutic or prophylactic efficacy is increased.For example, the engineered CAR or TCR expressed by the population canbe conjugated either directly or indirectly through a linker to atargeting moiety. The practice of conjugating compounds, e.g., the CARor TCR, to targeting moieties is known in the art. See, for instance,Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Pat. No.5,087,616.

III. Dosing

The timing and size of the multiple doses of cells generally aredesigned to reduce risk of or minimize toxic outcomes and/or to improveefficacy, such as by providing increased exposure of the subject to thecells, e.g., over time. The methods involve administering a first dose,generally followed by one or more consecutive doses, with particulartime frames between the different doses.

In the context of adoptive cell therapy, administration of a given“dose” encompasses administration of the given amount or number of cellsas a single composition and/or single uninterrupted administration,e.g., as a single injection or continuous infusion, and also encompassesadministration of the given amount or number of cells as a split dose,provided in multiple individual compositions or infusions, over aspecified period of time, which is no more than 3 days. Thus, in somecontexts, the first or consecutive dose is a single or continuousadministration of the specified number of cells, given or initiated at asingle point in time. In some contexts, however, the first orconsecutive dose is administered in multiple injections or infusionsover a period of no more than three days, such as once a day for threedays or for two days or by multiple infusions over a single day period.

Thus, in some aspects, the cells of the first dose are administered in asingle pharmaceutical composition. In some embodiments, the cells of theconsecutive dose are administered in a single pharmaceuticalcomposition.

In some embodiments, the cells of the first dose are administered in aplurality of compositions, collectively containing the cells of thefirst dose. In some embodiments, the cells of the consecutive dose areadministered in a plurality of compositions, collectively containing thecells of the consecutive dose. In some aspects, additional consecutivedoses may be administered in a plurality of compositions over a periodof no more than 3 days.

The term “split dose” refers to a dose that is split so that it isadministered over more than one day. This type of dosing is encompassedby the present methods and is considered to be a single dose.

Thus, the first dose and/or consecutive dose(s) in some aspects may beadministered as a split dose. For example, in some embodiments, the dosemay be administered to the subject over 2 days or over 3 days. Exemplarymethods for split dosing include administering 25% of the dose on thefirst day and administering the remaining 75% of the dose on the secondday. In other embodiments, 33% of the first dose may be administered onthe first day and the remaining 67% administered on the second day. Insome aspects, 10% of the dose is administered on the first day, 30% ofthe dose is administered on the second day, and 60% of the dose isadministered on the third day. In some embodiments, the split dose isnot spread over more than 3 days.

With reference to a prior dose, such as a first dose, the term“consecutive dose” refers to a dose that is administered to the samesubject after the prior, e.g., first, dose without any intervening doseshaving been administered to the subject in the interim. Nonetheless, theterm does not encompass the second, third, and/or so forth, injection orinfusion in a series of infusions or injections comprised within asingle split dose. Thus, unless otherwise specified, a second infusionwithin a one, two or three-day period is not considered to be a“consecutive” dose as used herein. Likewise, a second, third, andso-forth in the series of multiple doses within a split dose also is notconsidered to be an “intervening” dose in the context of the meaning of“consecutive” dose. Thus, unless otherwise specified, a doseadministered a certain period of time, greater than three days, afterthe initiation of a first or prior dose, is considered to be a“consecutive” dose even if the subject received a second or subsequentinjection or infusion of the cells following the initiation of the firstdose, so long as the second or subsequent injection or infusion occurredwithin the three-day period following the initiation of the first orprior dose.

Thus, unless otherwise specified, multiple administrations of the samecells over a period of up to 3 days is considered to be a single dose,and administration of cells within 3 days of an initial administrationis not considered a consecutive dose and is not considered to be anintervening dose for purposes of determining whether a second dose is“consecutive” to the first.

In some embodiments, multiple consecutive doses are given, in someaspects using the same timing guidelines as those with respect to thetiming between the first dose and first consecutive dose, e.g., byadministering a first and multiple consecutive doses, with eachconsecutive dose given within a period of time that is greater thanabout 14 and less than about 28 days, e.g., about 21 days, after theadministration of the first or immediately prior dose. The additionalmultiple additional consecutive dose or doses also are referred to assubsequent dose or subsequent consecutive dose.

As used herein, “first dose” is used to describe the timing of a givendose being prior to the administration of a consecutive or subsequentdose. The term does not necessarily imply that the subject has neverbefore received a dose of cell therapy or even that the subject has notbefore received a dose of the same cells or cells expressing the samerecombinant receptor or targeting the same antigen.

Dosage Amount or Size

The size of the first and/or one or more consecutive doses of cells aregenerally designed to provide improved efficacy and/or reduced risk oftoxicity. In some embodiments, the number of cells in the first dose isbetween about 0.5×10⁶ cells/kg body weight of the subject and 3×10⁶cells/kg, between about 0.75×10⁶ cells/kg and 2.5×10⁶ cells/kg orbetween about 1×10⁶ cells/kg and 2×10⁶ cells/kg, each inclusive.

In some embodiments, the first dose is a low dose. In particularembodiments, the first dose contains a number of cells, number ofrecombinant receptor (e.g., CAR)-expressing cells, number of T cells, ornumber of peripheral blood mononuclear cells (PBMCs) in the range fromabout 10⁵ to about 10⁶ of such cells per kilogram body weight of thesubject, inclusive, and/or a number of such cells that is no more thanabout 10⁵ or about 10⁶ such cells per kilogram body weight of thesubject, inclusive. For example, in some embodiments, the first doseincludes less than or no more than at or about 1×10⁵, at or about 2×10⁵,at or about 5×10⁵, or at or about 1×10⁶ of such cells per kilogram bodyweight of the subject. In some embodiments, the first dose includes ator about 1×10⁵, at or about 2×10⁵, at or about 5×10⁵, or at or about1×10⁶ of such cells per kilogram body weight of the subject, or a valuewithin the range between any two of the foregoing values. In particularembodiments, the numbers and/or concentrations of cells refer to thenumber of recombinant receptor (e.g., CAR)-expressing cells. In otherembodiments, the numbers and/or concentrations of cells refer to thenumber or concentration of all cells, T cells, or peripheral bloodmononuclear cells (PBMCs) administered.

In some embodiments, for example, where the subject is a human, thefirst dose includes fewer than or equal to about 1×10⁸ total recombinantreceptor (e.g., CAR)-expressing cells, T cells, or peripheral bloodmononuclear cells (PBMCs), e.g., in the range of about 1×10⁶ to 1×10⁸such cells, inclusive, such as no more than 2×10⁶, 5×10⁶, 1×10⁷, 5×10⁷,or 1×10⁸ or total such cells, or the range between any two of theforegoing values.

In some embodiments, the first dose contains fewer than or equal toabout 1×10⁸ total recombinant receptor (e.g., CAR)-expressing cells, Tcells, or peripheral blood mononuclear cells (PBMCs) cells per m² of thesubject, e.g., in the range of about 1×10⁶ to 1×10⁸ such cells per m² ofthe subject, inclusive, such as no more than 2×10⁶, 5×10⁶, 1×10⁷, 5×10⁷,or 1×10⁸ such cells per m² of the subject, or the range between any twoof the foregoing values.

In certain embodiments, for example, where risk of toxicity and/ordisease burden in the subject is determined to be low, the first dosecan be a relatively high dose, such as a dose that is greater than 1×10⁶cells/kg or that includes greater than 1×10⁸ cells, such as T cells orPBMCs, or greater than 1×10⁸ such cells per m² of the subject. In someembodiments, disease burden is low if the subject does not exhibitsubstantial morphologic disease or does not exhibit morphologic disease,or that exhibits less than 20% of blast cells in bone marrow, less than15% of blast cells in bone marrow, less than 10% blast cells in bonemarrow or less than 5% blast cells in bone marrow. In some embodiments,disease burden is low if the subject exhibits non-morphologic disease,such as exhibits minimal residual disease or molecularly detectabledisease but does not exhibit the features associated with morphologicaldisease as known in the art of described elsewhere, such as does notexhibit greater than 5% of blast cells in bone marrow. In someembodiments, the number of cells, recombinant receptor (e.g.,CAR)-expressing cells, T cells, or peripheral blood mononuclear cells(PBMCs) in the first dose is greater than about 1×10⁶ such cells perkilogram body weight of the subject, e.g., 2×10⁶, 3×10⁶, 5×10⁶, 1×10⁷,5×10⁷, 1×10⁸, 1×10⁹, or 1×10¹⁰ such cells per kilogram of body weightand/or 1×10⁸, 1×10⁹, 1×10¹⁰ such cells per m² of the subject or total,or the range between any two of the foregoing values.

In some embodiments, the number of cells administered in the consecutivedose is the same as or similar to the number of cells administered inthe first dose in any of the embodiments herein, such as less than or nomore than at or about 1×10⁵, at or about 2×10⁵, at or about 5×10⁵, or ator about 1×10⁶ of such cells per kilogram body weight of the subject. Insome embodiments, the consecutive dose(s) contains at or about 1×10⁵, ator about 2×10⁵, at or about 5×10⁵, or at or about 1×10⁶ of such cellsper kilogram body weight of the subject, or a value within the rangebetween any two of the foregoing values.

In reference to cell numbers, in some embodiments, such values refer tonumbers of recombinant receptor-expressing (e.g. CAR-expressing) cells;in other embodiments, they refer to number of T cells or PBMCs or totalcells administered.

In some aspects, the consecutive dose is larger than the first dose. Forexample, in some embodiments, the consecutive dose contains more thanabout 1×10⁶ cells, recombinant receptor (e.g. CAR)-expressing cells, Tcells, and/or PBMCs per kilogram body weight of the subject, such asabout or at least about 2×10⁶, 3×10⁶, 5×10⁶, 1×10⁷, 1×10⁸, or 1×10⁹ suchcells per kilogram body weight of the subject. In some embodiments, thenumber of cells in the consecutive dose is between about 2×10⁶ cells/kgbody weight of the subject and 6×10⁶ cells/kg, between about 2.5×10⁶cells/kg and 5.0×10⁶ cells/kg, or between about 3.0×10⁶ cells/kg andabout 4.0×10⁶ cells/kg, each inclusive. In some embodiments, the amountor size of the consecutive dose is sufficient to reduce disease burdenor an indicator thereof, and/or one or more symptoms of the disease orcondition. In some embodiments, the dose is of a size effective toimprove survival of the subject, for example, to induce survival,relapse-free survival, or event-free survival of the subject for atleast 6 months, or at least 1, 2, 3, 4, or 5 years. In some embodiments,the number of cells, recombinant receptor (e.g. CAR)-expressing cells, Tcells, and/or PBMCs administered and/or number of such cellsadministered per body weight of the subject in the consecutive dose isat least 2-fold, 5-fold, 10-fold, 50-fold, or 100-fold or more greaterthan the number administered in the first dose. In some embodiments,disease burden, tumor size, tumor volume, tumor mass, and/or tumor loador bulk is reduced following the consecutive dose by at least at orabout 50, 60, 70, 80, 90% or more compared to that immediately prior tothe administration of the first dose or of the consecutive dose.

In other embodiments, the number of cells administered in theconsecutive dose is lower than the number of cells administered in thefirst dose.

In some embodiments, multiple consecutive doses are administeredfollowing the first dose, such that an additional dose or doses areadministered following administration of the consecutive dose. In someaspects, the number of cells administered to the subject in theadditional dose or doses (i.e., the third, fourth, fifth, and so forth)is the same as or similar to the first dose and/or consecutive dose. Insome embodiments, the additional dose or doses are larger than priordoses.

In some aspects, the size of the first and/or consecutive dose isdetermined based on one or more criteria such as response of the subjectto prior treatment, e.g. chemotherapy, disease burden in the subject,such as tumor load, bulk, size, or degree, extent, or type ofmetastasis, stage, and/or likelihood or incidence of the subjectdeveloping toxic outcomes, e.g., CRS, macrophage activation syndrome,tumor lysis syndrome, neurotoxicity, and/or a host immune responseagainst the cells and/or recombinant receptors being administered.

In some aspects, the size of the first and/or consecutive dose isdetermined by the burden of the disease or condition in the subject. Forexample, in some aspects, the number of cells administered in the firstdose is determined based on the tumor burden that is present in thesubject immediately prior to administration of the first dose. In someembodiments, the size of the first and/or consecutive dose is inverselycorrelated with disease burden. In some aspects, as in the context of alarge disease burden, the subject is administered a low number of cells,for example less than about 1×10⁶ cells per kilogram of body weight ofthe subject. In other embodiments, as in the context of a lower diseaseburden, the subject is administered a larger number of cells, such asmore than about 1×10⁶ cells per kilogram body weight of the subject, forexample more than about 2×10⁶, 2.5×10⁶ or 3×10⁶ cells/kg.

In some aspects, the number of cells administered in the consecutivedose is determined based on the tumor burden that is present in thesubject following administration of the first dose. In some embodiments,e.g. where the first dose results in reduced or decreased disease burdenor has done so below a particular threshold amount or level, e.g., oneabove which there is an increased risk of toxic outcome, the consecutivedose is high or large, e.g. more than 1×10⁶ cells (e.g., total cells,receptor-expressing cells, T cells, or PBMCs) per kilogram body weight,such as more than 2.0×10⁶, 2.5×10⁶ or 3.0×10⁶ cells/kg, and/or is largerthan the first dose. In some embodiments, a subject exhibits reduced ordecreased disease burden if they exhibited morphological disease priorto treatment and exhibit complete remission (e.g., fewer than 5% blastsin bone marrow) with or without molecular disease (e.g., minimumresidual disease (MRD) that is molecularly detectable, e.g., as detectedby flow cytometry or quantitative PCR) after treatment. In someembodiments, a subject exhibits reduced or decreased disease burden ifthey exhibited molecular disease prior to treatment and do not exhibitmolecular disease after treatment.

In other aspects, the number of cells administered in the consecutivedose is low, e.g. less than about 1×10⁶, e.g. the same as or lower thanthe first dose, where the first dose has reduced tumor burden to a smallextent or where the first dose has not led to a detectable reduction intumor burden. In some cases, even if tumor burden is not reduced in asubject after receiving a first dose, a consecutive dose can be high orlarge, e.g. more than 1×10⁶ cells (e.g., total cells,receptor-expressing cells, T cells, or PBMCs) per kilogram body weight,such as more than 2.0×10⁶, 2.5×10⁶ or 3.0×10⁶ cells/kg, and/or is largerthan the first dose.

In some embodiments, the first dose includes the cells in an amount thatdoes not cause or reduces the likelihood of toxicity or toxic outcomes,such as cytokine release syndrome (CRS), severe CRS (sCRS), macrophageactivation syndrome, tumor lysis syndrome, fever of at least at or about38 degrees Celsius for three or more days and a plasma level of CRP ofat least at or about 20 mg/dL, neurotoxicity and/or neurotoxicity. Insome aspects, the number of cells administered in the first dose isdetermined based on the likelihood that the subject will exhibittoxicity or toxic outcomes, such as CRS, sCRS, and/or CRS-relatedoutcomes following administration of the cells. For example, in someembodiments, the likelihood for the development of toxic outcomes in asubject is predicted based on tumor burden. In some embodiments, themethods include detecting or assessing the toxic outcome and/or diseaseburden prior to the administration of the dose.

In some aspects, the number of cells administered in the consecutivedose is determined based on the level of toxicity or toxic outcomes,e.g. CRS-related outcomes, following administration of the first dose.For example, in some embodiments, the number of cells administered inthe consecutive dose is low, e.g. less than 1×10⁶ cells per kilogrambody weight, e.g. the same as or lower than the first dose, if thesubject exhibits a detectable level of toxicity or toxic outcomes, e.g.CRS-related outcomes, following administration of the first dose.

In some embodiments, the subject is not administered the consecutivedose at a time following the first dose at which they exhibit toxicityor toxic outcomes, such as CRS-related outcomes, e.g. if a serum levelof an indicator of CRS or other biochemical indicator of the toxicity ismore than at or about 10 times, more than at or about 15 times, morethan at or about 20 times, more than at or about 25 times, more than ator about 50 times, more than at or about 75 times, more than at or about100 times, more than at or about 125 times, more than at or about 150times, more than at or about 200 times, or more than at or about 250times the baseline or pre-treatment level, such as the serum level ofthe indicator immediately prior to administration of the first dose.

In some embodiments, the subject is administered the consecutive dose,if and when, following the first dose, a biochemical indicator of orother indicator of a toxic outcome, e.g. a serum level of an indicatorof CRS has not increased to above a given level, e.g., an acceptablelevel, such as more than at or about 10, 15, 20, 25, 50, 75, or 100times the serum level of the indicator immediately prior toadministration of the first dose, or has increased above an acceptablelevel but has declined to at or below the acceptable level. In someaspects, the consecutive dose is administered if and when the indicatordeclines below the acceptable level within 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,or 35 days, e.g., does so within 21 days, and/or within 15-27 days,within 21 days, within 28 days, or within 35 days, of the administrationof the first dose, but is not administered if the level of the indicatordoes not decline below the acceptable level within that time period.

In some embodiments, the consecutive dose is administered if and when aclinical risk for developing cytokine-release syndrome (CRS), macrophageactivation syndrome, or tumor lysis syndrome, or neurotoxicity is notpresent or has passed or has subsided following the firstadministration, such as after a critical window after which such eventsgenerally have subsided and/or are less likely to occur, e.g., in 60,70, 80, 90, or 95% of subjects with a particular disease or condition.

In some embodiments, whether a consecutive dose is administered, whenthe consecutive dose is administered, and/or the number of the cellsadministered in the consecutive dose is or are determined based on thepresence, absence, or degree of an immune response or detectable immuneresponse in the subject to the cells of the first dose or recombinantreceptor expressed thereby. In some aspects, a consecutive dosecontaining cells expressing the receptor of the cells of the first dosewill not be administered to a subject with a detectable host adaptiveimmune response, or an immune response that has become established orreached a certain level, stage, or degree.

Timing of Doses

In some aspects, the timing of the consecutive dose is measured from theinitiation of the first dose to the initiation of the consecutive dose.In other embodiments, the timing of the consecutive dose is measuredfrom the completion of the first dose, or from the median day ofadministration of the first dose, e.g. in the context of split dosing,described herein, where a dose is administered over more than one day,e.g. over 2 days or over 3 days.

In some embodiments, the consecutive dose is administered at a time atwhich a serum level of a factor indicative of CRS in the subject is nomore than 10 times, 25 times, 50 times, or 100 times the serum level ofsaid indicator in the subject immediately prior to the administration ofthe first dose.

In some embodiments, the consecutive dose is administered at a timeafter a CRS-related outcome, such as a serum factor associated with orindicative of CRS, or a clinical sign or symptom thereof such as fever,hypoxia, hypotension, or neurological disturbance, in the subject hasreached a peak level and begun to decline following administration ofthe first dose. In some embodiments, the consecutive dose isadministered at a time after which the outcome is observed to be on thedecline compared with the highest level of such outcome measuredfollowing the administration, or at a time at which or after which thelevel is on the decline following the maximum value or level of theoutcome reached after the administration.

In some embodiments, the consecutive dose is administered when the levelof an indicator of a toxic outcome, such as a serum indicator of CRS,declines below 25 times the level of the indicator immediately prior tothe first dose. In some aspects, the consecutive dose is administered ata time at which the subject does not exhibit CRS or does not exhibitsevere CRS.

In some aspects, the consecutive dose is administered at a point in timeat which the disease burden in the patient has decreased as compared tothe disease burden immediately prior to administration of the firstdose. In some embodiments, a subject exhibits reduced or decreaseddisease burden if they exhibited morphological disease prior totreatment and exhibit complete remission (e.g., fewer than 5% blasts inbone marrow) with or without molecular disease (e.g., minimum residualdisease (MRD) that is molecularly detectable, e.g., as detected by flowcytometry or quantitative PCR) after treatment. In some embodiments, asubject exhibits reduced or decreased disease burden if they exhibitedmolecular disease prior to treatment and do not exhibit moleculardisease after treatment. In some embodiments, the consecutive dose isadministered at a time at which the disease burden or indicator thereofsuch as bulk or number or percentage of disease (e.g., tumor) cells inthe blood, other fluid, organ or tissue of the subject, or size of thetumor, has decreased by 10, 20, 30, 40, 50, 60, 70, 80, or 90% or morefollowing administration of the first dose.

In some embodiments, the consecutive dose is administered at a point intime at which the disease or condition in the subject has not relapsedfollowing the reduction in response to the first or prior dose. In someembodiments, the disease burden reduction is indicated by a reduction inone or more factors, such as load or number of disease cells in thesubject or fluid or organ or tissue thereof, the mass or volume of atumor, or the degree or extent of metastases. Such a factor is deemed tohave relapsed if after reduction in the factor in response to an initialtreatment or administration, the factor subsequently increases. In someembodiments, the relapse is in one or one or more factors, or in thedisease burden generally. In some aspects, the consecutive dose isadministered at a point in time at which the subject, disease burden, orfactor thereof has relapsed as compared to the lowest point measured orreached following the first or prior administration, but still is lowercompared to the time immediately prior to the first dose. In someembodiments, the subject is administered the consecutive dose at a pointin time at which disease burden or factor indicative thereof has notchanged, e.g. at a time when an increase in disease burden has beenprevented.

In some embodiments, the consecutive dose is administered at a time whena host adaptive immune response is not detected, has not becomeestablished, or has not reached a certain level, degree, or stage. Insome aspects, the consecutive dose is administered prior to thedevelopment of a memory immune response in the subject.

In some aspects, the time between the administration of the first doseand the administration of the consecutive dose is about 9 to about 35days, about 14 to about 28 days, or 15 to 27 days. In some embodiments,the administration of the consecutive dose is at a time point more thanabout 14 days after and less than about 28 days after the administrationof the first dose. In some embodiments, the administration of theconsecutive dose is no more than about 14 days, 15 days, 16 days, 17days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25days, 26 days, 27 days or 28 days after the administration of the firstdose. In some aspects, the time between the first and consecutive doseis about 14 days about 17 days or about 21 days.

In some embodiments, an additional or subsequent dose or doses, e.g.further consecutive doses, are administered following administration ofthe consecutive dose. In some aspects, the additional consecutive doseor doses are administered at least about 14 and less than about 28 daysfollowing administration of a prior dose, such as a prior consecutivedose. In some embodiments, an exemplary dosage regime includes aschedule of administration of recombinant receptor-expressing cells(e.g., CAR-expressing cells, such as CAR-expressing T cells) at or aboutIn some embodiments, the additional dose is administered less than about14 days following the prior dose, for example, less than 4, 5, 6, 7, 8,9, 10, 11, 12, or 13 days after the prior dose. In some embodiments, nodose is administered less than about 14 days following the prior doseand/or no dose is administered more than about 28 days after the priordose.

In any of the embodiments, the methods in some cases include theadministration of the first or prior dose and the consecutive dose(s),and in other cases include the administration of the consecutive dose(s)to a subject who has previously received the first or prior dose but donot include the administration of the first or prior dose itself. Thus,the methods in some cases involve the administration of consolidatingtreatment, such as by administering a consolidating consecutive dose toa subject that has previously received a dose, e.g., a debulking dose,of recombinant receptor-expressing, e.g., CAR-expressing, cells. In someaspects, the previous dose of receptor-expressing, e.g.,CAR,-expressing, cells has been sufficient to reduce the burden of thedisease or condition in the subject such that the efficacy and/or safetyof the administration of the cells in the consecutive dose is improvedrelative to administering the dose without the subject having receivedthe first dose.

In some embodiments, disease burden, tumor size, tumor volume, tumormass, and/or tumor load or bulk is reduced following the consecutivedose by at least at or about 50, 60, 70, 80, 90% or more compared tothat immediately prior to the administration of the first dose or of theconsecutive dose.

IV. Toxicity and Toxic Outcomes

In some embodiments, the timing or amount of the doses reduces orprevents toxicity or an outcome or symptom thereof, for example,compared to administration of the same or similar total number of cellsas a single-dose, administration of the consecutive dose without thesubject having previously received the first dose, administration of theconsecutive dose at a different time relative to the first dose, and/oradministration of different amount(s) in one or more of the doses.

Administration of adoptive T cell therapy, such as treatment with Tcells expressing chimeric antigen receptors, can induce toxic effects oroutcomes such as cytokine release syndrome and neurotoxicity. In someexamples, such effects or outcomes parallel high levels of circulatingcytokines, which may underlie the observed toxicity.

In some embodiments, the provided methods are designed to or includefeatures that result in a lower degree of toxicity, toxic outcome orsymptom, toxicity-promoting profile, factor, or property, such as asymptom or outcome associated with or indicative of cytokine releasesyndrome (CRS), for example, compared to administration of the same orsimilar total number of cells as a single-dose, administration of theconsecutive dose without the subject having previously received thefirst dose, administration of the consecutive dose at a different timerelative to the first dose, and/or administration of different amount(s)in one or more of the doses.

In some aspects, the toxic outcome is or is associated with orindicative of cytokine release syndrome (CRS) or severe CRS (sCRS). CRS,e.g., sCRS, can occur in some cases following adoptive T cell therapyand administration to subjects of other biological products. See Davilaet al., Sci Transl Med 6, 224ra25 (2014); Brentjens et al., Sci. Transl.Med. 5, 177ra38 (2013); Grupp et al., N. Engl. J. Med. 368, 1509-1518(2013); and Kochenderfer et al., Blood 119, 2709-2720 (2012); Xu et al.,Cancer Letters 343 (2014) 172-78.

Typically, CRS is caused by an exaggerated systemic immune responsemediated by, for example, T cells, B cells, NK cells, monocytes, and/ormacrophages. Such cells may release a large amount of inflammatorymediators such as cytokines and chemokines. Cytokines may trigger anacute inflammatory response and/or induce endothelial organ damage,which may result in microvascular leakage, heart failure, or death.Severe, life-threatening CRS can lead to pulmonary infiltration and lunginjury, renal failure, or disseminated intravascular coagulation. Othersevere, life-threatening toxicities can include cardiac toxicity,respiratory distress, neurologic toxicity and/or hepatic failure.

CRS may be treated using anti-inflammatory therapy such as an anti-IL-6therapy, e.g., anti-IL-6 antibody, e.g., tocilizumab, or antibiotics. Insome embodiments, the subject is treated with such a therapy followingthe first administration and the consecutive dose is administered onlyif and when the CRS-associated symptom(s) are reduced or declining ordeclined below an acceptable level following such treatment.

Outcomes, signs and symptoms of CRS are known and include thosedescribed herein. In some embodiments, where a particular dosage regimenor administration effects or does not effect a given CRS-associatedoutcome, sign, or symptom, particular outcomes, signs, and symptomsand/or quantities or degrees thereof may be specified.

In the context of administering CAR-expressing cells, CRS typicallyoccurs 6-20 days after infusion of cells that express a CAR. See Xu etal., Cancer Letters 343 (2014) 172-78. In some cases, CRS occurs lessthan 6 days or more than 20 days after CAR T cell infusion. Theincidence and timing of CRS may be related to baseline cytokine levelsor tumor burden at the time of infusion. Commonly, CRS involves elevatedserum levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α,and/or interleukin (IL)-2. Other cytokines that may be rapidly inducedin CRS are IL-1β, IL-6, IL-8, and IL-10.

In some aspects, a lower degree of toxicity, outcome, symptom, profile,factor, or property is observed in the subjects to which the cells areadministered by the dosing regimen of the present methods, for example,as compared to administration of the same or similar total number ofcells as a single-dose, administration of the consecutive dose withoutthe subject having previously received the first dose, administration ofthe consecutive dose at a different time relative to the first dose,and/or administration of different amount(s) in one or more of thedoses. For example, in some embodiments, following administration of thefirst dose to the subject, the subject exhibits a lower degree of aCRS-related outcome, and/or exhibits a lower serum level of aninflammatory cytokine or factor indicative of CRS, as compared toadministration of a larger number of cells to the subject. In someembodiments, following administration to the subject of the consecutivedose, the subject exhibits a lower degree of a CRS-related outcome,and/or exhibits a lower serum level of an inflammatory cytokine oroutcome indicative of CRS, as compared to administration of theconsecutive dose to the subject without the subject having received thefirst dose, or as compared to administering the first and consecutivedose(s) as a single dose, or as compared to administering theconsecutive dose at a time that is earlier than or after the specifiedtime period. In some embodiments, the subject does not exhibit CRS orsevere CRS following administration of the first dose and/or followingadministration of the consecutive dose.

Exemplary outcomes associated with CRS include fever, rigors, chills,hypotension, dyspnea, acute respiratory distress syndrome (ARDS),encephalopathy, ALT/AST elevation, renal failure, cardiac disorders,hypoxia, neurologic disturbances, and death. Neurological complicationsinclude delirium, seizure-like activity, confusion, word-findingdifficulty, aphasia, and/or becoming obtunded. Other CRS-relatedoutcomes include fatigue, nausea, headache, seizure, tachycardia,myalgias, rash, acute vascular leak syndrome, liver function impairment,and renal failure. In some aspects, CRS is associated with an increasein one or more factors such as serum-ferritin, d-dimer,aminotransferases, lactate dehydrogenase and triglycerides, or withhypofibrinogenemia or hepatosplenomegaly.

In some embodiments, outcomes associated with CRS include one or moreof: persistent fever, e.g., fever of a specified temperature, e.g.,greater than at or about 38 degrees Celsius, for two or more, e.g.,three or more, e.g., four or more days or for at least three consecutivedays; fever greater than at or about 38 degrees Celsius; elevation ofcytokines, such as a max fold change, e.g., of at least at or about 75,compared to pre-treatment levels of at least two cytokines (e.g., atleast two of the group consisting of interferon gamma (IFNγ), GM-CSF,IL-6, IL-10, Flt-3L, fracktalkine, and IL-5, and/or tumor necrosisfactor alpha (TNFα)), or a max fold change, e.g., of at least at orabout 250 of at least one of such cytokines; and/or at least oneclinical sign of toxicity, such as hypotension (e.g., as measured by atleast one intravenous vasoactive pressor); hypoxia (e.g., plasma oxygen(PO₂) levels of less than at or about 90%); and/or one or moreneurologic disorders (including mental status changes, obtundation, andseizures).

Exemplary CRS-related outcomes include increased or high serum levels ofone or more factors, including cytokines and chemokines and otherfactors associated with CRS. Exemplary outcomes further includeincreases in synthesis or secretion of one or more of such factors. Suchsynthesis or secretion can be by the T cell or a cell that interactswith the T cell, such as an innate immune cell or B cell.

In some embodiments, the CRS-associated serum factors or CRS-relatedoutcomes include inflammatory cytokines and/or chemokines, includinginterferon gamma (IFN-γ), TNF-α, IL-1β, IL-2, IL-6, IL-7, IL-8, IL-10,IL-12, sIL-2Ra, granulocyte macrophage colony stimulating factor(GM-CSF), macrophage inflammatory protein (MIP)-1, tumor necrosis factoralpha (TNFα), IL-6, and IL-10, IL-1β, IL-8, IL-2, MIP-1, Flt-3L,fracktalkine, and/or IL-5. In some embodiments, the factor or outcomeincludes C reactive protein (CRP). In addition to being an early andeasily measurable risk factor for CRS, CRP also is a marker for cellexpansion. In some embodiments, subjects that are measured to have highlevels of CRP, such as ≧15 mg/dL, have CRS. In some embodiments,subjects that are measured to have high levels of CRP do not have CRS.In some embodiments, a measure of CRS includes a measure of CRP andanother factor indicative of CRS.

In some embodiments, one or more inflammatory cytokines or chemokinesare monitored before, during, or after CAR treatment. In some aspects,the one or more cytokines or chemokines include IFN-γ, TNF-α, IL-2,IL-1β, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Rα, granulocyte macrophagecolony stimulating factor (GM-CSF), or macrophage inflammatory protein(MIP). In some embodiments, IFN-γ, TNF-α, and IL-6 are monitored.

CRS criteria that appear to correlate with the onset of CRS to predictwhich patients are more likely to be at risk for developing sCRS havebeen developed (see Davilla et al. Science translational medicine. 2014;6(224):224ra25). Factors include fevers, hypoxia, hypotension,neurologic changes, elevated serum levels of inflammatory cytokines,such as a set of seven cytokines (IFNγ, IL-5, IL-6, IL-10, Flt-3L,fractalkine, and GM-CSF) whose treatment-induced elevation can correlatewell with both pretreatment tumor burden and sCRS symptoms. Otherguidelines on the diagnosis and management of CRS are known (see e.g.,Lee et al, Blood. 2014; 124(2):188-95). In some embodiments, thecriteria reflective of CRS grade are those detailed in Table 1 below.

TABLE 1 Exemplary Grading Criteria for CRS Grade Description of Symptoms1 Not life-threatening, require only symptomatic Mild treatment such asantipyretics and anti-emetics (e.g., fever, nausea, fatigue, headache,myalgias, malaise) 2 Require and respond to moderate intervention:Moderate Oxygen requirement <40%, or Hypotension responsive to fluids orlow dose of a single vasopressor, or Grade 2 organ toxicity (by CTCAEv4.0) 3 Require and respond to aggressive intervention: Severe Oxygenrequirement ≧40%, or Hypotension requiring high dose of a singlevasopressor (e.g., norepinephrine ≧20 μg/kg/min, dopamine ≧10 μg/kg/min,phenylephrine ≧200 μg/kg/min, or epinephrine ≧ 10 μg/kg/min), orHypotension requiring multiple vasopressors (e.g., vasopressin + one ofthe above agents, or combination vasopressors equivalent to ≧20μg/kg/min norepinephrine), or Grade 3 organ toxicity or Grade 4transaminitis (by CTCAE v4.0) 4 Life-threatening: Life-threateningRequirement for ventilator support, or Grade 4 organ toxicity (excludingtransaminitis) 5 Death Fatal

As used herein, a subject is deemed to develop “severe CRS” (“sCRS”) inresponse to or secondary to administration of a cell therapy or dose ofcells thereof, if, following administration, the subject displays: (1)fever of at least 38 degrees Celsius for at least three days; (2)cytokine elevation that includes either (a) a max fold change of atleast 75 for at least two of the following group of seven cytokinescompared to the level immediately following the administration:interferon gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fracktalkine, andIL-5 and/or (b) a max fold change of at least 250 for at least one ofthe following group of seven cytokines compared to the level immediatelyfollowing the administration: interferon gamma (IFNγ), GM-CSF, IL-6,IL-10, Flt-3L, fracktalkine, and IL-5; and (c) at least one clinicalsign of toxicity such as hypotension (requiring at least one intravenousvasoactive pressor) or hypoxia (PO₂<90%) or one or more neurologicdisorder(s) (including mental status changes, obtundation, and/orseizures). In some embodiments, severe CRS includes CRS with a grade of3 or greater, such as set forth in Table 1.

In some embodiments, the CRS encompasses a combination of (1) persistentfever (fever of at least 38 degrees Celsius for at least three days) and(2) a serum level of CRP of at least at or about 20 mg/dL.

In some embodiments, the CRS encompasses hypotension requiring the useof two or more vasopressors or respiratory failure requiring mechanicalventilation.

The method of measuring or detecting the various outcomes may bespecified.

In some aspects, prior to the administration of the first dose,subsequent to the administration of the first dose and beforeadministration of the consecutive dose, or subsequent to theadministration of the consecutive dose, a CRS-associated outcome isassessed in the subject. In some embodiments, the level of the toxicoutcome, e.g. the CRS-related outcome, e.g. the serum level of anindicator of CRS, is measured by ELISA. In some embodiments, feverand/or levels of CRP can be measured. In some embodiments, subjects witha fever and a CRP≧15 mg/dL may be considered high-risk for developingsevere CRS.

In some embodiments, the toxic outcome, toxicity, or symptom is measuredat a specified time point following administration. In some embodiments,the toxic outcome, e.g. CRS, severe CRS, and/or the CRS-associatedoutcome or serum level, or the lower degree of the outcome or serumlevel, is measured at 24 hours, at day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 following theadministration, or over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, CRPis measured at day 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the foldchange in a factor or multiple factors, such as a cytokine(s), ismeasured as fold change between the level prior to treatment and at day2, 3, 4, 5, 6, 7, 14, or 20 or 21, following treatment.

In some embodiments, at the time of the administration of theconsecutive dose, the level of the CRS-related outcome is no more than50% of the peak level, is no more than 40% of the peak level, is no morethan 30% of the peak level, is no more than 20% of the peak level, is nomore than 15% of the peak level, is no more than 10% of the peak level,is no more than 5% of the peak level, or is at or about or below thelevel immediately prior to the administration of the first dose and/oris at or about or below baseline.

In some aspects, at the time of the administration of the consecutivedose, the level of the CRS-related outcome is no more than ten times thelevel immediately prior to the administration of the first dose.

In some embodiments, the CRS-related outcome in the subject at day 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 following the administration ofthe consecutive dose is not detectable or is reduced as compared to amethod where the subject is administered the consecutive dose withouthaving been administered the first dose and/or a method in which thecells of the first and second doses are administered in a single dose,and/or a method in which the consecutive dose is given at a time whichis earlier than the time between the first and consecutive dosesspecified by the method.

In some embodiments, the CRS-related outcome in the subject at day 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 following the administration ofthe first dose is not detectable or is reduced as compared to thatfollowing administration to the subject a dose of 2-fold, 5-fold,10-fold, or 100-fold the number of cells, T cells, cells expressing therecombinant receptor, or PBMCs.

In some embodiments, the area under the curve (AUC) for a CRS-relatedoutcome or serum level of a factor indicative of CRS over time in thesubject following administration of the consecutive dose is lower ascompared to that of a method where the subject is administered theconsecutive dose without having been administered the first dose, and/ora method in which the cells of the first and consecutive doses areadministered in a single dose, and/or a method in which the consecutivedose is given at a time which is earlier than the time between the firstand consecutive doses specified by the method.

In some embodiments, the area under the curve (AUC) for a CRS-relatedoutcome or serum level of a factor indicative of CRS over time in thesubject following administration of the first dose is lower as comparedto that following administration to the subject dose of 2-fold, 5-fold,10-fold, or 100-fold the number of cells, T cells, cells expressing therecombinant receptor, or PBMCs.

In some aspects, the toxic outcome is or is associated withneurotoxicity. In some embodiments, symptoms associated with a clinicalrisk of neurotoxicity include confusion, delirium, expressive aphasia,obtundation, myoclonus, lethargy, altered mental status, convulsions,seizure-like activity, seizures (optionally as confirmed byelectroencephalogram [EEG]), elevated levels of beta amyloid (Aβ),elevated levels of glutamate, and elevated levels of oxygen radicals. Insome embodiments, neurotoxicity is graded based on severity (e.g., usinga Grade 1-5 scale (see, e.g., Guido Cavaletti & Paola Marmiroli NatureReviews Neurology 6, 657-666 (December 2010); National CancerInstitute—Common Toxicity Criteria version 4.03 (NCI-CTCAE v4.03).

In some instances, neurologic symptoms may be the earliest symptoms ofsCRS. In some embodiments, neurologic symptoms are seen to begin 5 to 7days after cell therapy infusion. In some embodiments, duration ofneurologic changes may range from 3 to 19 days. In some cases, recoveryof neurologic changes occurs after other symptoms of sCRS have resolved.In some embodiments, time or degree of resolution of neurologic changesis not hastened by treatment with anti-IL-6 and/or steroid(s).

As used herein, a subject is deemed to develop “severe neurotoxicity” inresponse to or secondary to administration of a cell therapy or dose ofcells thereof, if, following administration, the subject displayssymptoms that limit self-care (e.g. bathing, dressing and undressing,feeding, using the toilet, taking medications) from among: 1) symptomsof peripheral motor neuropathy, including inflammation or degenerationof the peripheral motor nerves; 2) symptoms of peripheral sensoryneuropathy, including inflammation or degeneration of the peripheralsensory nerves, dysesthesia, such as distortion of sensory perception,resulting in an abnormal and unpleasant sensation, neuralgia, such asintense painful sensation along a nerve or a group of nerves, and/orparesthesia, such as functional disturbances of sensory neuronsresulting in abnormal cutaneous sensations of tingling, numbness,pressure, cold and warmth in the absence of stimulus. In someembodiments, severe neurotoxicity includes neurotoxicity with a grade of3 or greater, such as set forth in Table 2.

TABLE 2 Exemplary Grading Criteria for neurotoxicity Grade Descriptionof Symptoms 1 Mild or asymptomatic symptoms Asymptomatic or Mild 2Presence of symptoms that limit instrumental Moderate activities ofdaily living (ADL),such as preparing meals, shopping for groceries orclothes, using the telephone, managing money 3 Presence of symptoms thatlimit self-care ADL, Severe such as bathing, dressing and undressing,feeding self, using the toilet, taking medications 4 Symptoms that arelife-threatening, requiring Life-threatening urgent intervention 5 DeathFatal

In some embodiments, the methods reduce symptoms associated withneurotoxicity compared to other methods. For example, subjects treatedaccording to the present methods may have reduced symptoms ofneurotoxicity, such as limb weakness or numbness, loss of memory,vision, and/or intellect, uncontrollable obsessive and/or compulsivebehaviors, delusions, headache, cognitive and behavioral problemsincluding loss of motor control, cognitive deterioration, and autonomicnervous system dysfunction, and sexual dysfunction, compared to subjectstreated by other methods. In some embodiments, subjects treatedaccording to the present methods may have reduced symptoms associatedwith peripheral motor neuropathy, peripheral sensory neuropathy,dysethesia, neuralgia or paresthesia.

In some embodiments, the methods reduce outcomes associated withneurotoxicity including damages to the nervous system and/or brain, suchas the death of neurons. In some aspects, the methods reduce the levelof factors associated with neurotoxicity such as beta amyloid (Aβ),glutamate, and oxygen radicals.

In some embodiments, subjects administered the consecutive dosefollowing the first dose have reduced symptoms, outcomes, or factorsassociated with neurotoxicity compared to administration of theconsecutive dose in the absence of the first dose, administration of thecells of the first and second doses in a single dose, and/oradministration of the consecutive dose at a time which is earlier thanthe time between the first and consecutive doses specified by themethod.

V. Host Immune Responses to Transferred Cells

In some embodiments, one or more of the doses, e.g., the consecutivedose(s), is administered at a time at which an immune response, e.g., anadaptive or specific immune response to the transgenic receptor orcells, in the subject is not present, not detectable, or not detectableabove a certain level. The presence or degree of a specific immuneresponse to the transgene generally is related to the immunogenicity ofthe receptor, e.g., the CAR or transgenic TCR, expressed by the cells,and/or the time during which the subject has been exposed to the cells.For example, in some embodiments, an immune response, e.g., a specifichumoral and/or cell-mediated immune response against the receptor, isnot detected before 28 days, 35 days, or 42 days following the firstexposure of the subject to the cells expressing the receptor. Thus, insome embodiments, the consecutive dose is administered before an immuneresponse, an adaptive or specific immune response, a detectable immuneresponse, and/or a memory response against the recombinant receptor orcells has developed in the subject. In this regard, the ability of cellsof the consecutive dose to expand and/or persist in the subject isimproved in comparison to other methods in which a consecutive dose isgiven at a later time point in comparison with the prior or first dose.

The methods may involve the detection of the presence or absence orlevel of such an immune response or indicator thereof, for example,following the administration of a first or consecutive dose and beforethe administration of the consecutive or next consecutive dose.

In some embodiments, the decision of when and/or whether to administerthe consecutive dose depends on whether the subject exhibits such animmune response or detectable readout thereof, e.g., a detectablespecific or adaptive host immune response specific for the cells orrecombinant receptor, e.g., CAR, expressed by the cells of the firstdose, and/or whether such a response is detected above a certain level.In some embodiments, where such a response is detected, the subject isnot administered the consecutive dose.

In general, the consecutive dose is administered at a time at which thesubject does not exhibit a specific or adaptive, e.g., humoral orcell-mediated, immune response against the receptor, e.g., CAR,expressed by the cells of the first dose, or does not exhibit such aresponse or indicator thereof at a detectable level or above anacceptable level. In some aspects, at the time of administration of theconsecutive dose, the subject exhibits a reduced humoral orcell-mediated immune response against the CAR expressed by the cells ofthe first dose as compared to when an initial dose is larger.

In some embodiments, the host immune response is or comprises a humoralimmune response. The humoral immune response may be indicated by thepresence of antibodies specific for the cells or receptors expressedthereby in the serum, other bodily fluid, and/or organ or tissue of thesubject. In some embodiments, such antibodies of a particular isotypeare present, such as IgM or IgG, e.g., IgG1, IgG2, IgG3, and/or IgG4; insome embodiments they include IgE.

In some embodiments, the immune response is or comprises a cell-mediatedcomponent. A cell-mediated response may be indicated by the presence ofcells, e.g., T cells, e.g., helper or cytotoxic T cells, thatspecifically recognize one or more epitopes of the recombinant receptoror cells via a T cell receptor.

In some embodiments the immune response is a primary immune response; insome aspects, the immune response is a memory response.

In some of any of the above embodiments, a detectable immune responserefers to an amount detectable by any of a number of known methods forassessing specific immune responses to particular antigens and cells.For example, in some embodiments, the immune response of the specifiedtype is detectable by performing ELISpot, ELISAs, or cell-based antibodydetection methods, for example, by flow cytometry, on serum from thesubject to detect the presence of antibodies that specifically bind toand/or neutralize antigens present on the cells, e.g., binding toepitopes of the recombinant receptor, e.g., CAR. In some such assays,isotype of the detected antibody is determined and may indicate the typeof response and/or whether the response is a memory response.

In some embodiments, the specified immune response is detectable bycytotoxic T-lymphocyte (CTL) assays for detection of CD8⁺ T cells thatspecifically bind to and induce cytotoxicity in response to epitopes inthe recombinant receptor, and/or a mixed lymphocyte reaction, usingcells, e.g., irradiated cells, expressing the recombinant receptor, asstimulator cells.

In some aspects, the detectable immune response is one that is detectedby such a method above or significantly above the level of a controlsample, such as a non-coated well or well coated with a control peptideor cells not expressing the recombinant receptor and/or levels detectedbased on pre-treatment serum or blood sample from the subject prior totreatment with the cells expressing the recombinant receptors.

In some aspects, the presence or absence of such a host immune responseand/or quantity, degree, or extent thereof, is detected or measured, forexample, following the administration of the first dose or consecutivedose.

Humoral immune responses may be detected by any of a number ofwell-known assays for detection of antibodies specific for particularantigens or cells, including binding assays, immunoassays, andcell-based assays. The assays may include those designed to assess thepresence or absence of particular functions of the antibodies, such astheir ability to carry out a particular effector function upon bindingto the antigen, such as neutralizing antibody assays. In someembodiments, outcomes of humoral immune responses, such asantigen-specific antibodies, e.g., neutralizing antibodies, are detectedusing cell-based assays, e.g., by incubating pre- and post-treatmentcells from the subject with cells expressing the recombinant receptor(and control cells) and detecting antigen-specific binding and/or otheroutcomes, such as neutralizing outcomes, e.g., by flow cytometry orenzymatic assays. In some embodiments, ELISA, and/or ELISpot assays areused to detect and quantify antibodies specific for the recombinantreceptors, such as CARs, and epitopes mapped using known techniques,such as those using individual peptides representing portions of thereceptor. See, e.g., Jensen et al. Biol Blood Marrow Transplant. 2010September; 16(9): 1245-1256. In some embodiments, isotype of thedetected antibodies are assessed, for example by using detectionantibodies specific for particular isotypes, e.g., human isotypes.

Cellular or cell-based immune response to the cells and/or receptors maybe detected and/or measured using any of a number of well-knowntechniques. Such techniques may include cytotoxic T-lymphocyte (CTL)assays for detection of CD8⁺ T cells that specifically bind to andinduce cytotoxicity in response to epitopes in the recombinant receptor,e.g., CAR, and/or cells administered. In some embodiments, the assay isa mixed lymphocyte reaction, such as those using PBMCs or otherhost-derived cells from blood or other organ or tissue as respondercells, and cells induced to express the recombinant receptor, e.g.,irradiated T cells expressing the CAR, as stimulator cells. Thestimulator cells generally are autologous and may be the same cellsadministered to the subject, and may be irradiated. Non-transduced cellsor cells not expressing the transgene of interest may be used asnegative controls in place of the stimulator cells in control samples.Likewise, responder cell samples from pre-treatment time points or othersubjects may be used in control samples. In some aspects, such assaysassess the ability of host cells to carry out one or more effectorfunctions, e.g., antigen-specific cell lysis, e.g., using a chromiumrelease assay to detect cytotoxic T cells present in the subject whichspecifically recognize antigens present on or in the administered cellsand induce a cytotoxic response. In some embodiments, peripheral bloodcells, e.g., PBMCs, are obtained from a subject before and afteradministration of the cells, and each used in an assay, such as a celllysis assay, using autologous T cells modified to express therecombinant receptor, which generally are irradiated. Specific lysisindicates the presence of receptor-specific cell-mediated immuneresponse. Epitope mapping may be carried out using panels of peptidesrepresenting portions of the recombinant receptor. See, e.g., Riddell etal., Nature Medicine 2, 216-223 (1996); Lamers, Blood 2011 117: 72-82.HLA tetramer binding assays may be used for the enumeration ofantigen-specific T cells. In some aspects, lymphoproliferative assays(LPAs) and/or assays to assess for secreted cytokines, such as ELISAsand/or intracellular staining and assessment by flow cytometry, are usedfor detection of transgene-specific CD4+ T cells.

In some embodiments, the method prevents the induction of or reduces thelevel of antibodies against the receptor, e.g. anti-CAR antibodies. Forexample, antibody titers of anti-receptor, e.g. anti-CAR, antibodies,for example, as measured in the serum of the subject by ELISA, aredecreased following administration of the consecutive dose, as comparedto methods in which a consecutive dose is administered at a differenttime relative to the administration of the first dose, such as at alater time, e.g., following relapse. Thus, in some embodiments, themethods improve efficacy by increasing exposure of the subject to theadministered cells by preventing or reducing host immune responses thatwould otherwise clear or prevent expansion of the administered cells.

VI. Disease Burden

The administration, e.g., of one or more of the doses, generally reducesor prevents the expansion or burden of the disease or condition in thesubject. For example, where the disease or condition is a tumor, themethods generally reduce tumor size, bulk, metastasis, percentage ofblasts in the bone marrow or molecularly detectable cancer and/orimprove prognosis or survival or other symptom associated with tumorburden. In some embodiments, administration of the consecutive dose istimed with respect to a decrease in burden and/or relapse following thefirst or prior dose.

Disease burden can encompass a total number of cells of the disease inthe subject or in an organ, tissue, or bodily fluid of the subject, suchas the organ or tissue of the tumor or another location, e.g., whichwould indicate metastasis. For example, tumor cells may be detectedand/or quantified in the blood or bone marrow in the context of certainhematological malignancies. Disease burden can include, in someembodiments, the mass of a tumor, the number or extent of metastasesand/or the percentage of blast cells present in the bone marrow.

In some embodiments, a subject has leukemia. The extent of diseaseburden can be determined by assessment of residual leukemia in blood orbone marrow. In some embodiments, a subject exhibits morphologic diseaseif there are greater than or equal to 5% blasts in the bone marrow, forexample, as detected by light microscopy. In some embodiments, a subjectexhibits complete or clinical remission if there are less than 5% blastsin the bone marrow.

In some embodiments, a subject may exhibit complete remission, but asmall proportion of morphologically undetectable (by light microscopytechniques) residual leukemic cells are present. A subject is said toexhibit minimum residual disease (MRD) if the subject exhibits less than5% blasts in the bone marrow and exhibits molecularly detectable cancer.In some embodiments, molecularly detectable cancer can be assessed usingany of a variety of molecular techniques that permit sensitive detectionof a small number of cells. In some aspects, such techniques include PCRassays, which can determine unique Ig/T-cell receptor generearrangements or fusion transcripts produced by chromosometranslocations. In some embodiments, flow cytometry can be used toidentify cancer cell based on leukemia-specific immunophenotypes. Insome embodiments, molecular detection of cancer can detect as few as 1leukemia cell in 100,000 normal cells. In some embodiments, a subjectexhibits MRD that is molecularly detectable if at least or greater than1 leukemia cell in 100,000 cells is detected, such as by PCR or flowcytometry. In some embodiments, the disease burden of a subject ismolecularly undetectable or MRD⁻, such that, in some cases, no leukemiacells are able to be detected in the subject using PCR or flow cytometrytechniques.

In some embodiments, the methods and/or administration of the first dosedecrease(s) disease burden as compared with disease burden at a timeimmediately prior to the administration of the first dose. In someaspects, administration of the first dose reduces disease burden, e.g.tumor burden. In some embodiments, the consecutive dose effects areduction, e.g., a further reduction, in disease burden.

In some embodiments, the first dose contains the cells in an amount thatis effective to reduce burden of a disease or condition in the subject,e.g., tumor burden. In some embodiments, e.g. where the disease orcondition is a tumor, administration of the first dose is one thatdebulks the tumor. As used herein, a “debulking dose” refers to a dosethat is effective to at least partially reduce burden of the disease orcondition, e.g. tumor burden, in the subject. In some aspects, thedebulking dose may not completely eradicate the disease or condition.

In some aspects, administration of the first dose and/or consecutivedose may prevent an increase in disease burden, and this may beevidenced by no change in disease burden.

In some aspects, the disease or condition persists followingadministration of the first dose and/or administration of the first doseis not sufficient to eradicate the disease or condition in the subject.

In some aspects, administration of the consecutive dose reduces diseaseburden as compared to disease burden at a time immediately prior to thefirst dose, or at a time immediately prior to the consecutive dose. Insome aspects, for example in the context of relapse, administration ofthe consecutive dose effects a reduction in disease burden as comparedto the peak level of disease burden following administration of thefirst dose.

In some embodiments, the method reduces the burden of the disease orcondition, e.g., number of tumor cells, size of tumor, duration ofpatient survival or event-free survival, to a greater degree and/or fora greater period of time as compared to the reduction that would beobserved with a comparable method using an alternative dosing regimen,such as one in which the subject receives a single dose, e.g., a singlelarge dose, of cells, e.g. administration of the total number of cellsadministered in the first dose and the consecutive dose, collectively,in the provided methods, instead as a single dose, or administration ofmultiple large doses or multiple doses spaced from one another by lessthan about 14 or more than about 28 days. In some embodiments, diseaseburden is reduced to a greater extent or for a greater durationfollowing the consecutive dose compared to the reduction that would beeffected by administering the consecutive dose to a subject having notreceived the first dose.

In some embodiments, the burden of a disease or condition in the subjectis detected, assessed, or measured. Disease burden may be detected insome aspects by detecting the total number of disease ordisease-associated cells, e.g., tumor cells, in the subject, or in anorgan, tissue, or bodily fluid of the subject, such as blood or serum.In some embodiments, disease burden, e.g. tumor burden, is assessed bymeasuring the mass of a solid tumor and/or the number or extent ofmetastases. In some aspects, survival of the subject, survival within acertain time period, extent of survival, presence or duration ofevent-free or symptom-free survival, or relapse-free survival, isassessed. In some embodiments, any symptom of the disease or conditionis assessed. In some embodiments, the measure of disease or conditionburden is specified.

In some aspects, disease burden is measured or detected prior toadministration of the first dose, following the administration of thefirst dose but prior to administration of the consecutive dose, and/orfollowing administration of the consecutive dose. In the context ofmultiple consecutive doses, disease burden in some embodiments may bemeasured prior to or following any of the consecutive doses, or at atime between administration of consecutive doses.

In some embodiments, the burden is decreased by or by at least at orabout 10, 20, 30, 40, 50, 60, 70, 90, or 100 percent afteradministration of the first dose. In some aspects, administration of theconsecutive dose effects a further reduction in disease burden, e.g.tumor burden, such as a at or about 10, 20, 30, 40, 50, 60, 70, 90, or100 percent decrease in burden compared to immediately prior to theadministration of the consecutive dose or overall compared toimmediately prior to the first dose. In some embodiments, diseaseburden, tumor size, tumor volume, tumor mass, and/or tumor load or bulkis reduced following the consecutive dose by at least at or about 50,60, 70, 80, 90% or more compared to that immediately prior to theadministration of the first dose or of the consecutive dose.

In some embodiments, reduction of disease burden by the method comprisesan induction in morphologic complete remission, for example, as assessedat 1 month, 2 months, 3 months, or more than 3 months, afteradministration of, e.g., initiation of, the first or consecutive dose.In some aspects, an assay for minimal residual disease, for example, asmeasured by multiparametric flow cytometry, is negative, or the level ofminimal residual disease is less than about 0.3%, less than about 0.2%,less than about 0.1%, or less than about 0.05%.

In some embodiments, the event-free survival rate or overall survivalrate of the subject is improved by the methods, as compared with othermethods. For example, in some embodiments, event-free survival rate orprobability for subjects treated by the methods at 6 months followingthe first dose is greater than about 40%, greater than about 50%,greater than about 60%, greater than about 70%, greater than about 80%,greater than about 90%, or greater than about 95%. In some aspects,overall survival rate is greater than about 40%, greater than about 50%,greater than about 60%, greater than about 70%, greater than about 80%,greater than about 90%, or greater than about 95%. In some embodiments,the subject treated with the methods exhibits event-free survival,relapse-free survival, or survival to at least 6 months, or at least 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, the time toprogression is improved, such as a time to progression of greater thanat or about 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10years.

In some embodiments, following treatment by the method, the probabilityof relapse is reduced as compared to other methods. For example, in someembodiments, the probability of relapse at 6 months following the firstdose is less than about 80%, less than about 70%, less than about 60%,less than about 50%, less than about 40%, less than about 30%, less thanabout 20%, or less than about 10%.

In some aspects, reduction of disease burden, e.g. debulking of thetumor, following the first dose reduces toxicity or toxic outcomesfollowing the consecutive dose. Toxic outcomes following a reduction intumor burden can be assessed as described herein.

In some aspects, reduction of disease burden, e.g. debulking of thetumor, resulting from the present methods improves the persistence ofthe cells in the subject. For example, in some aspects, administrationof the first dose reduces disease burden, e.g. tumor burden, such thatthe cells administered in the consecutive dose persist for longer thancells administered by other dosing regimens, such as administering theconsecutive dose to a subject that has not been administered the cellsof the first dose.

VII. Cell Exposure and Persistence

In some embodiments, the dose amount(s) and/or timing thereof aredesigned to promote exposure of the subject to the cells, such as bypromoting their expansion and/or persistence over time.

In some embodiments, the provided methods increase exposure of thesubject to the administered cells (e.g., increased number of cells orduration over time) and/or improve efficacy and therapeutic outcomes inadoptive cell therapy. In some aspects, the methods are advantageous inthat a greater and/or longer degree of exposure to the cells expressingthe recombinant receptors, e.g., CAR-expressing cells, improvestreatment outcomes as compared with other methods. Such outcomes mayinclude patient survival and remission, even in individuals with severetumor burden.

In some embodiments, the administration of the first dose, e.g., firstlow dose, increases maximum, total, and/or duration of exposure to thecells in the subject as compared to administration of a high initialdose of the cells. In some aspects, administration of the first dose inthe context of high disease burden (and thus higher amounts of antigen)enhances efficacy as compared with administration of a larger dose inthe same context, which may result in exhaustion which may preventexpansion and/or persistence of the cells. In some embodiments,administering the first dose in the context of high disease burdenreduces exhaustion of the transferred cells, thereby increasing clinicalefficacy as compared to other methods, such as those where a higherinitial dose is administered.

In some embodiments, the presence and/or amount of cells expressing therecombinant receptor (e.g., CAR-expressing cells) in the subjectfollowing the first dose and/or following the consecutive dose isdetected. In some aspects, quantitative PCR (qPCR) is used to assess thequantity of cells expressing the recombinant receptor (e.g.,CAR-expressing cells) in the blood or serum or organ or tissue (e.g.,disease site) of the subject. In some aspects, persistence is quantifiedas copies of DNA or plasmid encoding the receptor, e.g., CAR, permicrogram of DNA, or as the number of receptor-expressing, e.g.,CAR-expressing, cells per microliter of the sample, e.g., of blood orserum, or per total number of peripheral blood mononuclear cells (PBMCs)or white blood cells or T cells per microliter of the sample.

In some embodiments, the cells are detected in the subject at or atleast at 4, 14, 15, 27, or 28 days following the administration of thefirst dose. In some aspects, the cells are detected at or at least at 2,4, or 6 weeks following, or 3, 6, or 12, 18, or 24, or 30 or 36 months,or 1, 2, 3, 4, 5, or more years, following administration of the firstor consecutive dose.

In some embodiments, the persistence of receptor, e.g., CAR, -expressingcells in the subject by the methods, following the consecutive dose,and/or following administration of the first dose, is greater ascompared to that which would be achieved by alternative methods such asthose involving the administration of a single dose, e.g., containing alarger number of cells than the first dose, administration of the cellsof the collective doses as a single dose, administration of the cells ofthe consecutive dose without the subject having received the first dose,and/or administration of the consecutive dose at a time that is outsidethe specified time window such as later than the time specified orfollowing the mounting of an immune response by the subject against thereceptor, e.g., the CAR.

In some embodiments, the persistence and/or expansion and/or presence ofrecombinant receptor-expressing, e.g., CAR-expressing, cells in thesubject following administration of the consecutive dose is greater ascompared to that achieved via a method using an alternative dosingregimen, such as one involving the administration of the cells of theconsecutive dose without the subject having been administered the cellsof the first dose or where the subject is administered the cellscollectively administered in the first and consecutive doses as a singledose.

The exposure, e.g., number of cells, indicative of expansion and/orpersistence, may be stated in terms of maximum numbers of the cells towhich the subject is exposed, duration of detectable cells or cellsabove a certain number or percentage, area under the curve for number ofcells over time, and/or combinations thereof and indicators thereof.Such outcomes may be assessed using known methods, such as qPCR todetect copy number of nucleic acid encoding the recombinant receptorcompared to total amount of nucleic acid or DNA in the particularsample, e.g., blood or serum, and/or flow cytometric assays detectingcells expressing the receptor generally using antibodies specific forthe receptors. Cell-based assays may also be used to detect the numberor percentage of functional cells, such as cells capable of binding toand/or neutralizing and/or inducing responses, e.g., cytotoxicresponses, against cells of the disease or condition or expressing theantigen recognized by the receptor.

In some aspects, increased exposure of the subject to the cells includesincreased expansion of the cells. In some embodiments, the receptor-(e.g., CAR-) expressing cells expand in the subject followingadministration of the first dose and/or following administration of theconsecutive dose. In some aspects, the methods result in greaterexpansion of the cells compared with other methods, such as thoseinvolving the administration of the cells as a single dose,administration of larger first doses, administration of the consecutivedose without administering the first dose, and/or methods in which aconsecutive dose is administered before or after the specified window oftime or time point, such that, for example, an immune response developsprior to the administration of the consecutive dose.

In some aspects, the method results in high in vivo proliferation of theadministered cells, for example, as measured by flow cytometry. In someaspects, high peak proportions of the cells are detected. For example,in some embodiments, at a peak or maximum level following the first orconsecutive administration, in the blood or disease-site of the subjector white blood cell fraction thereof, e.g., PBMC fraction or T cellfraction, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, or at least about 90% of the cells express therecombinant receptor, e.g., the CAR.

In some embodiments, the method results in a maximum concentration, inthe blood or serum or other bodily fluid or organ or tissue of thesubject, of at least 100, 500, 1000, 1500, 2000, 5000, 10,000 or 15,000copies of or nucleic acid encoding the receptor, e.g., the CAR permicrogram of DNA, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or0.9 receptor-expressing, e.g., CAR,-expressing cells per total number ofperipheral blood mononuclear cells (PBMCs), total number of mononuclearcells, total number of T cells, or total number of microliters. In someembodiments, the cells expressing the receptor are detected as at least10, 20, 30, 40, 50, or 60% of total PBMCs in the blood of the subject,and/or at such a level for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 24, 36, 48, or 52 weeks following the first or consecutiveadministration or for 1, 2, 3, 4, or 5, or more years following suchadministration.

In some aspects, the method results in at least a 2-fold, at least a4-fold, at least a 10-fold, or at least a 20-fold increase in copies ofnucleic acid encoding the recombinant receptor, e.g., CAR, per microgramof DNA, e.g., in the serum of the subject.

In some embodiments, cells expressing the receptor are detectable in theblood or serum of the subject, e.g., by a specified method, such as qPCRor flow cytometry-based detection method, at least 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or60 or more days following administration of the first dose or afteradministration of the consecutive dose, for at least at or about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 or more weeks following the administration of the first doseor the consecutive dose.

In some aspects, at least about 1×10², at least about 1×10³, at leastabout 1×10⁴, at least about 1×10⁵, or at least about 1×10⁶ or at leastabout 5×10⁶ or at least about 1×10⁷ or at least about 5×10⁷ or at leastabout 1×10⁸ recombinant receptor-expressing, e.g., CAR-expressing cells,and/or at least 10, 25, 50, 100, 200, 300, 400, or 500, or 1000receptor-expressing cells per microliter, e.g., at least 10 permicroliter, are detectable or are present in the subject or fluid,tissue, or compartment thereof, such as in the blood, e.g., peripheralblood, or disease site thereof. In some embodiments, such a number orconcentration of cells is detectable in the subject for at least about20 days, at least about 40 days, or at least about 60 days, or at leastabout 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 2 or 3years, following administration of the first dose or following theadministration of the consecutive dose(s). Such cell numbers may be asdetected by flow cytometry-based or quantitative PCR-based methods andextrapolation to total cell numbers using known methods. See, e.g.,Brentjens et al., Sci Transl Med. 2013 5(177), Park et al, MolecularTherapy 15(4):825-833 (2007), Savoldo et al., JCI 121(5):1822-1826(2011), Davila et al. (2013) PLoS ONE 8(4):e61338, Davila et al.,Oncoimmunology 1(9):1577-1583 (2012), Lamers, Blood 2011 117:72-82,Jensen et al. Biol Blood Marrow Transplant 2010 September; 16(9):1245-1256, Brentjens et al., Blood 2011 118(18):4817-4828.

In some aspects, the copy number of nucleic acid encoding therecombinant receptor, e.g., vector copy number, per 100 cells, forexample in the peripheral blood or bone marrow or other compartment, asmeasured by immunohistochemistry, PCR, and/or flow cytometry, is atleast 0.01, at least 0.1, at least 1, or at least 10, at about 1 week,about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or at leastabout 6 weeks, or at least about 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, or 12months or at least 2 or 3 years following administration of the cells,e.g., the first or consecutive dose(s). In some embodiments, the copynumber of the vector expressing the receptor, e.g. CAR, per microgram ofgenomic DNA is at least 100, at least 1000, at least 5000, or at least10,000, or at least 15,000 or at least 20,000 at a time about 1 week,about 2 weeks, about 3 weeks, or at least about 4 weeks followingadministration of the first dose or consecutive dose(s) ofreceptor-expressing, e.g. CAR-expressing, cells, or at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12 months or at least 2 or 3 years following suchadministration.

In some aspects, the receptor, e.g. CAR, expressed by the cells, isdetectable by quantitative PCR (qPCR) or by flow cytometry in thesubject, blood thereof, and/or disease site thereof, at a time that isat least about 3 months, at least about 6 months, at least about 12months, at least about 1 year, at least about 2 years, at least about 3years, or more than 3 years, following the administration of the cells,e.g., following the initiation of the administration of the first doseor the consecutive dose or subsequent consecutive dose.

In some embodiments, the area under the curve (AUC) for concentration ofreceptor- (e.g., CAR-) expressing cells in a fluid, tissue, or organ,e.g., blood, of the subject over time following the administration ofthe first dose is greater as compared to that achieved via analternative dosing regimen where the subject is administered the cellsof the first dose and the consecutive dose as a single dose.

In some aspects, the area under the curve (AUC) for concentration ofreceptor- (e.g., CAR-) expressing cells in a fluid, tissue, or organ,e.g., blood, of the subject over time following the administration ofthe consecutive dose is greater as compared to that achieved via analternative dosing regimen where the subject is administered theconsecutive dose without having been administered the first dose or inwhich the cells of the first and second doses are administered in asingle dose.

VIII. Recombinant Receptors Expressed by the Cells

The cells generally express recombinant receptors, including antigenreceptors such as functional non-TCR antigen receptors, e.g., chimericantigen receptors (CARs), and other antigen-binding receptors such astransgenic T cell receptors (TCRs). Also among the receptors are otherchimeric receptors.

Exemplary antigen receptors, including CARs, and methods for engineeringand introducing such receptors into cells, include those described, forexample, in international patent application publication numbersWO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321,WO2013/071154, WO2013/123061 U.S. patent application publication numbersUS2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995,7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319,7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118,and European patent application number EP2537416, and/or those describedby Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila etal. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol.,2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2):160-75. In some aspects, the antigen receptors include a CAR asdescribed in U.S. Pat. No. 7,446,190, and those described inInternational Patent Application Publication No.: WO/2014055668 A1.Examples of the CARs include CARs as disclosed in any of theaforementioned publications, such as WO2014031687, U.S. Pat. No.8,339,645, U.S. Pat. No. 7,446,179, US 2013/0149337, U.S. Pat. No.7,446,190, U.S. Pat. No. 8,389,282, Kochenderfer et al., 2013, NatureReviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J.Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 20135(177). See also WO2014031687, U.S. Pat. No. 8,339,645, U.S. Pat. No.7,446,179, US 2013/0149337, U.S. Pat. No. 7,446,190, and U.S. Pat. No.8,389,282.

Among the chimeric receptors are chimeric antigen receptors (CARs). Thechimeric receptors, such as CARs, generally include an extracellularantigen binding domain, such as a portion of an antibody molecule,generally a variable heavy (V_(H)) chain region and/or variable light(V_(L)) chain region of the antibody, e.g., an scFv antibody fragment.

In some embodiments, the antibody portion of the recombinant receptor,e.g., CAR, further includes a spacer, which may be or include at least aportion of an immunoglobulin constant region or variant or modifiedversion thereof, such as a hinge region, e.g., an IgG4 hinge region,and/or a CH1/CL and/or Fc region. In some embodiments, the constantregion or portion is of a human IgG, such as IgG4 or IgG1. In someaspects, the portion of the constant region serves as a spacer regionbetween the antigen-recognition component, e.g., scFv, and transmembranedomain. The spacer can be of a length that provides for increasedresponsiveness of the cell following antigen binding, as compared to inthe absence of the spacer. In some examples, the spacer is or is about12 amino acids in length or is no more than 12 amino acids in length.Exemplary spacers include those having at least about 10 to 229 aminoacids, about 10 to 200 amino acids, about 10 to 175 amino acids, about10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids,about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20amino acids, or about 10 to 15 amino acids, and including any integerbetween the endpoints of any of the listed ranges. In some embodiments,a spacer region has about 12 amino acids or less, about 119 amino acidsor less, or about 229 amino acids or less. Exemplary spacers includeIgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4hinge linked to the CH3 domain. Exemplary spacers include, but are notlimited to, those described in Hudecek et al. (2013) Clin. Cancer Res.,19:3153, international patent application publication numberWO2014031687, U.S. Pat. No. 8,822,647 or published app. No.US2014/0271635.

In some embodiments, the constant region or portion is of a human IgG,such as IgG4 or IgG1. In some embodiments, the spacer has the sequenceESKYGPPCPPCP (set forth in SEQ ID NO: 1), and is encoded by the sequenceset forth in SEQ ID NO: 2. In some embodiments, the spacer has thesequence set forth in SEQ ID NO: 3. In some embodiments, the spacer hasthe sequence set forth in SEQ ID NO: 4. In some embodiments, theconstant region or portion is of IgD. In some embodiments, the spacerhas the sequence set forth in SEQ ID NO: 5. In some embodiments, thespacer has a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to any of SEQ ID NOS: 1, 3, 4 or 5.

This antigen recognition domain generally is linked to one or moreintracellular signaling components, such as signaling components thatmimic activation through an antigen receptor complex, such as a TCRcomplex, in the case of a CAR, and/or signal via another cell surfacereceptor. Thus, in some embodiments, the antigen-binding component(e.g., antibody) is linked to one or more transmembrane andintracellular signaling domains. In some embodiments, the transmembranedomain is fused to the extracellular domain. In one embodiment, atransmembrane domain that naturally is associated with one of thedomains in the receptor, e.g., CAR, is used. In some instances, thetransmembrane domain is selected or modified by amino acid substitutionto avoid binding of such domains to the transmembrane domains of thesame or different surface membrane proteins to minimize interactionswith other members of the receptor complex.

The transmembrane domain in some embodiments is derived either from anatural or from a synthetic source. Where the source is natural, thedomain in some aspects is derived from any membrane-bound ortransmembrane protein. Transmembrane regions include those derived from(i.e. comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154 and/or transmembrane regions containing functional variants thereofsuch as those retaining a substantial portion of the structural, e.g.,transmembrane, properties thereof. In some embodiments, thetransmembrane domain is a transmembrane domain derived from CD4, CD28,or CD8, e.g., CD8alpha, or functional variant thereof. Alternatively thetransmembrane domain in some embodiments is synthetic. In some aspects,the synthetic transmembrane domain comprises predominantly hydrophobicresidues such as leucine and valine. In some aspects, a triplet ofphenylalanine, tryptophan and valine will be found at each end of asynthetic transmembrane domain. In some embodiments, the linkage is bylinkers, spacers, and/or transmembrane domain(s).

Among the intracellular signaling domains are those that mimic orapproximate a signal through a natural antigen receptor, a signalthrough such a receptor in combination with a costimulatory receptor,and/or a signal through a costimulatory receptor alone. In someembodiments, a short oligo- or polypeptide linker, for example, a linkerof between 2 and 10 amino acids in length, such as one containingglycines and serines, e.g., glycine-serine doublet, is present and formsa linkage between the transmembrane domain and the cytoplasmic signalingdomain of the CAR.

The receptor, e.g., the CAR, generally includes at least oneintracellular signaling component or components. In some embodiments,the receptor includes an intracellular component of a TCR complex, suchas a TCR CD3 chain that mediates T-cell activation and cytotoxicity,e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portionis linked to one or more cell signaling modules. In some embodiments,cell signaling modules include CD3 transmembrane domain, CD3intracellular signaling domains, and/or other CD transmembrane domains.In some embodiments, the receptor, e.g., CAR, further includes a portionof one or more additional molecules such as Fc receptor γ, CD8, CD4,CD25, or CD16. For example, in some aspects, the CAR or other chimericreceptor includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fcreceptor γ and CD8, CD4, CD25 or CD16.

In some embodiments, upon ligation of the CAR or other chimericreceptor, the cytoplasmic domain or intracellular signaling domain ofthe receptor activates at least one of the normal effector functions orresponses of the immune cell, e.g., T cell engineered to express theCAR. For example, in some contexts, the CAR induces a function of a Tcell such as cytolytic activity or T-helper activity, such as secretionof cytokines or other factors. In some embodiments, a truncated portionof an intracellular signaling domain of an antigen receptor component orcostimulatory molecule is used in place of an intact immunostimulatorychain, for example, if it transduces the effector function signal. Insome embodiments, the intracellular signaling domain or domains includethe cytoplasmic sequences of the T cell receptor (TCR), and in someaspects also those of co-receptors that in the natural context act inconcert with such receptors to initiate signal transduction followingantigen receptor engagement.

In the context of a natural TCR, full activation generally requires notonly signaling through the TCR, but also a costimulatory signal. Thus,in some embodiments, to promote full activation, a component forgenerating secondary or co-stimulatory signal is also included in theCAR. In other embodiments, the CAR does not include a component forgenerating a costimulatory signal. In some aspects, an additional CAR isexpressed in the same cell and provides the component for generating thesecondary or costimulatory signal.

T cell activation is in some aspects described as being mediated by twoclasses of cytoplasmic signaling sequences: those that initiateantigen-dependent primary activation through the TCR (primarycytoplasmic signaling sequences), and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences). In some aspects, theCAR includes one or both of such signaling components.

In some aspects, the CAR includes a primary cytoplasmic signalingsequence that regulates primary activation of the TCR complex. Primarycytoplasmic signaling sequences that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs. Examples of ITAM containingprimary cytoplasmic signaling sequences include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22,CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signalingmolecule(s) in the CAR contain(s) a cytoplasmic signaling domain,portion thereof, or sequence derived from CD3 zeta.

In some embodiments, the CAR includes a signaling domain and/ortransmembrane portion of a costimulatory receptor, such as CD28, 4-1BB,OX40, DAP10, and ICOS. In some aspects, the same CAR includes both theactivating and costimulatory components.

In some embodiments, the activating domain is included within one CAR,whereas the costimulatory component is provided by another CARrecognizing another antigen. In some embodiments, the CARs includeactivating or stimulatory CARs, costimulatory CARs, both expressed onthe same cell (see WO2014/055668). In some aspects, the cells includeone or more stimulatory or activating CAR and/or a costimulatory CAR. Insome embodiments, the cells further include inhibitory CARs (iCARs, seeFedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such asa CAR recognizing an antigen other than the one associated with and/orspecific for the disease or condition whereby an activating signaldelivered through the disease-targeting CAR is diminished or inhibitedby binding of the inhibitory CAR to its ligand, e.g., to reduceoff-target effects.

In some embodiments, the intracellular signaling component of therecombinant receptor, such as CAR, comprises a CD3 zeta intracellulardomain and a costimulatory signaling region. In certain embodiments, theintracellular signaling domain comprises a CD28 transmembrane andsignaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain.In some embodiments, the intracellular signaling domain comprises achimeric CD28 and/or CD137 (4-1BB, TNFRSF9) co-stimulatory domains,linked to a CD3 zeta intracellular domain.

In some embodiments, the CAR encompasses one or more, e.g., two or more,costimulatory domains and an activation domain, e.g., primary activationdomain, in the cytoplasmic portion. Exemplary CARs include intracellularcomponents of CD3-zeta, CD28, and 4-1BB.

In some embodiments, the CAR or other antigen receptor further includesa marker, such as a cell surface marker, which may be used to confirmtransduction or engineering of the cell to express the receptor, such asa truncated version of a cell surface receptor, such as truncated EGFR(tEGFR). In some aspects, the marker includes all or part (e.g.,truncated form) of CD34, a NGFR, or epidermal growth factor receptor(e.g., tEGFR). In some embodiments, the nucleic acid encoding the markeris operably linked to a polynucleotide encoding for a linker sequence,such as a cleavable linker sequence, e.g., T2A. For example, a marker,and optionally a linker sequence, can be any as disclosed in publishedpatent application No. WO2014031687. For example, the marker can be atruncated EGFR (tEGFR) that is, optionally, linked to a linker sequence,such as a T2A cleavable linker sequence. An exemplary polypeptide for atruncated EGFR (e.g. tEGFR) comprises the sequence of amino acids setforth in SEQ ID NO: 15 or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to SEQ ID NO: 15. An exemplary T2Alinker sequence comprises the sequence of amino acids set forth in SEQID NO: 14 or a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to SEQ ID NO: 14.

In some embodiments, the marker is a molecule, e.g., cell surfaceprotein, not naturally found on T cells or not naturally found on thesurface of T cells, or a portion thereof.

In some embodiments, the molecule is a non-self molecule, e.g., non-selfprotein, i.e., one that is not recognized as “self” by the immune systemof the host into which the cells will be adoptively transferred.

In some embodiments, the marker serves no therapeutic function and/orproduces no effect other than to be used as a marker for geneticengineering, e.g., for selecting cells successfully engineered. In otherembodiments, the marker may be a therapeutic molecule or moleculeotherwise exerting some desired effect, such as a ligand for a cell tobe encountered in vivo, such as a costimulatory or immune checkpointmolecule to enhance and/or dampen responses of the cells upon adoptivetransfer and encounter with ligand.

In some cases, CARs are referred to as first, second, and/or thirdgeneration CARs. In some aspects, a first generation CAR is one thatsolely provides a CD3-chain induced signal upon antigen binding; in someaspects, a second-generation CARs is one that provides such a signal andcostimulatory signal, such as one including an intracellular signalingdomain from a costimulatory receptor such as CD28 or CD137; in someaspects, a third generation CAR is one that includes multiplecostimulatory domains of different costimulatory receptors.

In some embodiments, the chimeric antigen receptor includes anextracellular portion containing an antibody or antibody fragment. Insome aspects, the chimeric antigen receptor includes an extracellularportion containing the antibody or fragment and an intracellularsignaling domain. In some embodiments, the antibody or fragment includesan scFv and the intracellular domain contains an ITAM. In some aspects,the intracellular signaling domain includes a signaling domain of a zetachain of a CD3-zeta (CD3ζ) chain. In some embodiments, the chimericantigen receptor includes a transmembrane domain linking theextracellular domain and the intracellular signaling domain. In someaspects, the transmembrane domain contains a transmembrane portion ofCD28. In some embodiments, the chimeric antigen receptor contains anintracellular domain of a T cell costimulatory molecule. Theextracellular domain and transmembrane domain can be linked directly orindirectly. In some embodiments, the extracellular domain andtransmembrane are linked by a spacer, such as any described herein. Insome embodiments, the receptor contains extracellular portion of themolecule from which the transmembrane domain is derived, such as a CD28extracellular portion. In some embodiments, the chimeric antigenreceptor contains an intracellular domain derived from a T cellcostimulatory molecule or a functional variant thereof, such as betweenthe transmembrane domain and intracellular signaling domain. In someaspects, the T cell costimulatory molecule is CD28 or 41BB.

For example, in some embodiments, the CAR contains an antibody, e.g., anantibody fragment, a transmembrane domain that is or contains atransmembrane portion of CD28 or a functional variant thereof, and anintracellular signaling domain containing a signaling portion of CD28 orfunctional variant thereof and a signaling portion of CD3 zeta orfunctional variant thereof. In some embodiments, the CAR contains anantibody, e.g., antibody fragment, a transmembrane domain that is orcontains a transmembrane portion of CD28 or a functional variantthereof, and an intracellular signaling domain containing a signalingportion of a 4-1BB or functional variant thereof and a signaling portionof CD3 zeta or functional variant thereof. In some such embodiments, thereceptor further includes a spacer containing a portion of an Igmolecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4hinge, such as a hinge-only spacer.

In some embodiments, the transmembrane domain of the recombinantreceptor, e.g., the CAR, is or includes a transmembrane domain of humanCD28 (e.g. Accession No. P01747.1) or variant thereof, such as atransmembrane domain that comprises the sequence of amino acids setforth in SEQ ID NO: 6 or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to SEQ ID NO: 6; in some embodiments,the transmembrane-domain containing portion of the recombinant receptorcomprises the sequence of amino acids set forth in SEQ ID NO: 7 or asequence of amino acids having at least at or about 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity thereto.

In some embodiments, the intracellular signaling component(s) of therecombinant receptor, e.g. the CAR, contains an intracellularcostimulatory signaling domain of human CD28 or a functional variant orportion thereof, such as a domain with an LL to GG substitution atpositions 186-187 of a native CD28 protein. For example, theintracellular signaling domain can comprise the sequence of amino acidsset forth in SEQ ID NO: 8 or 9 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8 or 9. Insome embodiments, the intracellular domain comprises an intracellularcostimulatory signaling domain of 4-1BB (e.g. (Accession No. Q07011.1)or functional variant or portion thereof, such as the sequence of aminoacids set forth in SEQ ID NO: 10 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10.

In some embodiments, the intracellular signaling domain of therecombinant receptor, e.g. the CAR, comprises a human CD3 zetastimulatory signaling domain or functional variant thereof, such as an112 AA cytoplasmic domain of isoform 3 of human CD3ζ (Accession No.:P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No.7,446,190 or U.S. Pat. No. 8,911,993. For example, in some embodiments,the intracellular signaling domain comprises the sequence of amino acidsas set forth in SEQ ID NO: 11, 12 or 13 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11, 12or 13.

In some aspects, the spacer contains only a hinge region of an IgG, suchas only a hinge of IgG4 or IgG1, such as the hinge only spacer set forthin SEQ ID NO: 1. In other embodiments, the spacer is or contains an Ighinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and/orCH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., anIgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ IDNO: 4. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 3.In some embodiments, the spacer is or comprises a glycine-serine richsequence or other flexible linker such as known flexible linkers.

For example, in some embodiments, the CAR includes an antibody such asan antibody fragment, including scFvs, a spacer, such as a spacercontaining a portion of an immunoglobulin molecule, such as a hingeregion and/or one or more constant regions of a heavy chain molecule,such as an Ig-hinge containing spacer, a transmembrane domain containingall or a portion of a CD28-derived transmembrane domain, a CD28-derivedintracellular signaling domain, and a CD3 zeta signaling domain. In someembodiments, the CAR includes an antibody or fragment, such as scFv, aspacer such as any of the Ig-hinge containing spacers, a CD28-derivedtransmembrane domain, a 4-1BB-derived intracellular signaling domain,and a CD3 zeta-derived signaling domain.

In some embodiments, such CAR constructs further includes a T2Aribosomal skip element and/or a tEGFR sequence, e.g., downstream of theCAR, such as set forth in SEQ ID NO: 14 and/or 15, respectively, or asequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to SEQ ID NO: 14 or 15.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Polypeptides, including the provided receptors and otherpolypeptides, e.g., linkers or peptides, may include amino acid residuesincluding natural and/or non-natural amino acid residues. The terms alsoinclude post-expression modifications of the polypeptide, for example,glycosylation, sialylation, acetylation, and phosphorylation. In someaspects, the polypeptides may contain modifications with respect to anative or natural sequence, as long as the protein maintains the desiredactivity. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the proteins or errors due to PCRamplification.

In some embodiments, the receptor, e.g., the CAR, expressed by the cellsin the consecutive dose contains at least one immunoreactive epitope asthe receptor, e.g., the CAR, expressed by the cells of the first dose.In some aspects, the receptor, e.g., the CAR, expressed by the cellsadministered in the consecutive dose is identical to the receptor, e.g.,the CAR, expressed by the first dose or is substantially identical tothe receptor, e.g., the CAR, expressed by the cells of administered inthe first dose.

The recombinant receptors, such as CARs, expressed by the cellsadministered to the subject in the various doses generally recognize orspecifically bind to a molecule that is expressed in, associated with,and/or specific for the disease or condition or cells thereof beingtreated. Upon specific binding to the molecule, e.g., antigen, thereceptor generally delivers an immunostimulatory signal, such as anITAM-transduced signal, into the cell, thereby promoting an immuneresponse targeted to the disease or condition. For example, in someembodiments, the cells in the first dose express a CAR that specificallybinds to an antigen expressed by a cell or tissue of the disease orcondition or associated with the disease or condition.

The receptor, e.g., the CAR, expressed by the cells in the consecutivedose(s) generally specifically binds to the same antigen as the CAR ofthe first dose and is often the same receptor or extremely similar tothe receptor in the cells of the first dose. In some embodiments, thereceptor on the cells in the consecutive dose(s) is the same as orshares a large degree of identity with the receptor in the cells of thefirst dose.

In some embodiments, the CAR expressed by the cells of the consecutivedose contains the same scFv, the same signaling domains, and/or the samejunctions as the CAR expressed by the cells of the first dose. In someembodiments, it further contains the same costimulatory, stimulatory,transmembrane, and/or other domains as that of the first dose. In someembodiments, one or more component of the CAR of the consecutive dose isdistinct from the CAR of the first dose.

IX. Engineered Cells

Among the cells expressing the receptors and administered by theprovided methods are engineered cells.

The cells generally are eukaryotic cells, such as mammalian cells, andtypically are human cells. In some embodiments, the cells are derivedfrom the blood, bone marrow, lymph, or lymphoid organs, are cells of theimmune system, such as cells of the innate or adaptive immunity, e.g.,myeloid or lymphoid cells, including lymphocytes, typically T cellsand/or NK cells. Other exemplary cells include stem cells, such asmultipotent and pluripotent stem cells, including induced pluripotentstem cells (iPSCs). The cells typically are primary cells, such as thoseisolated directly from a subject and/or isolated from a subject andfrozen. In some embodiments, the cells include one or more subsets of Tcells or other cell types, such as whole T cell populations, CD4+ cells,CD8+ cells, and subpopulations thereof, such as those defined byfunction, activation state, maturity, potential for differentiation,expansion, recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. With reference to the subject to be treated,the cells may be allogeneic and/or autologous. Among the methods includeoff-the-shelf methods. In some aspects, such as for off-the-shelftechnologies, the cells are pluripotent and/or multipotent, such as stemcells, such as induced pluripotent stem cells (iPSCs). In someembodiments, the methods include isolating cells from the subject,preparing, processing, culturing, and/or engineering them, andre-introducing them into the same subject, before or aftercryopreservation.

Among the sub-types and subpopulations of T cells and/or of CD4+ and/orof CD8+ T cells are naïve T (T_(N)) cells, effector T cells (T_(EFF)),memory T cells and sub-types thereof, such as stem cell memory T(T_(SCM)), central memory T (T_(CM)), effector memory T (T_(EM)), orterminally differentiated effector memory T cells, tumor-infiltratinglymphocytes (TIL), immature T cells, mature T cells, helper T cells,cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturallyoccurring and adaptive regulatory T (Treg) cells, helper T cells, suchas TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells,follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, the cells are natural killer (NK) cells. In someembodiments, the cells are monocytes or granulocytes, e.g., myeloidcells, macrophages, neutrophils, dendritic cells, mast cells,eosinophils, and/or basophils.

In some embodiments, the cells include one or more nucleic acidsintroduced via genetic engineering, and thereby express recombinant orgenetically engineered products of such nucleic acids. In someembodiments, the nucleic acids are heterologous, i.e., normally notpresent in a cell or sample obtained from the cell, such as one obtainedfrom another organism or cell, which for example, is not ordinarilyfound in the cell being engineered and/or an organism from which suchcell is derived. In some embodiments, the nucleic acids are notnaturally occurring, such as a nucleic acid not found in nature,including one comprising chimeric combinations of nucleic acids encodingvarious domains from multiple different cell types.

Vectors and Methods for Genetic Engineering

Also provided are methods, compositions, and kits, for producing thegenetically engineered cells expressing recombinant receptors. Thegenetic engineering generally involves introduction of a nucleic acidencoding the recombinant or engineered component into the cell, such asby retroviral transduction, transfection, or transformation.

In some embodiments, gene transfer is accomplished by first stimulatingthe cell, such as by combining it with a stimulus that induces aresponse such as proliferation, survival, and/or activation, e.g., asmeasured by expression of a cytokine or activation marker, followed bytransduction of the activated cells, and expansion in culture to numberssufficient for clinical applications.

In some contexts, overexpression of a stimulatory factor (for example, alymphokine or a cytokine) may be toxic to a subject. Thus, in somecontexts, the engineered cells include gene segments that cause thecells to be susceptible to negative selection in vivo, such as uponadministration in adoptive immunotherapy. For example in some aspects,the cells are engineered so that they can be eliminated as a result of achange in the in vivo condition of the subject to which they areadministered. The negative selectable phenotype may result from theinsertion of a gene that confers sensitivity to an administered agent,for example, a compound. Negative selectable genes include the Herpessimplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al.,Cell II:223, 1977) which confers ganciclovir sensitivity; the cellularhypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adeninephosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase,(Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

In some aspects, the cells further are engineered to promote expressionof cytokines or other factors. Various methods for the introduction ofgenetically engineered components, e.g., antigen receptors, e.g., CARs,are well known and may be used with the provided methods andcompositions. Exemplary methods include those for transfer of nucleicacids encoding the receptors, including via viral, e.g., retroviral orlentiviral, transduction, transposons, and electroporation.

In some embodiments, recombinant nucleic acids are transferred intocells using recombinant infectious virus particles, such as, e.g.,vectors derived from simian virus 40 (SV40), adenoviruses,adeno-associated virus (AAV). In some embodiments, recombinant nucleicacids are transferred into T cells using recombinant lentiviral vectorsor retroviral vectors, such as gamma-retroviral vectors (see, e.g.,Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25;Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al.(2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011November 29(11): 550-557.

In some embodiments, the retroviral vector has a long terminal repeatsequence (LTR), e.g., a retroviral vector derived from the Moloneymurine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV),murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV),spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Mostretroviral vectors are derived from murine retroviruses. In someembodiments, the retroviruses include those derived from any avian ormammalian cell source. The retroviruses typically are amphotropic,meaning that they are capable of infecting host cells of severalspecies, including humans. In one embodiment, the gene to be expressedreplaces the retroviral gag, pol and/or env sequences. A number ofillustrative retroviral systems have been described (e.g., U.S. Pat.Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989)BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14;Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993)Cur. Opin. Genet. Develop. 3:102-109.

Methods of lentiviral transduction are known. Exemplary methods aredescribed in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701;Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009)Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood.102(2): 497-505.

In some embodiments, recombinant nucleic acids are transferred into Tcells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In some embodiments, recombinant nucleic acids aretransferred into T cells via transposition (see, e.g., Manuri et al.(2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec TherNucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic materialin immune cells include calcium phosphate transfection (e.g., asdescribed in Current Protocols in Molecular Biology, John Wiley & Sons,New York. N.Y.), protoplast fusion, cationic liposome-mediatedtransfection; tungsten particle-facilitated microparticle bombardment(Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNAco-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other approaches and vectors for transfer of the nucleic acids encodingthe recombinant products are those described, e.g., in internationalpatent application, Publication No.: WO2014055668, and U.S. Pat. No.7,446,190.

Among additional nucleic acids, e.g., genes for introduction are thoseto improve the efficacy of therapy, such as by promoting viabilityand/or function of transferred cells; genes to provide a genetic markerfor selection and/or evaluation of the cells, such as to assess in vivosurvival or localization; genes to improve safety, for example, bymaking the cell susceptible to negative selection in vivo as describedby Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell etal., Human Gene Therapy 3:319-338 (1992); see also the publications ofPCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use ofbifunctional selectable fusion genes derived from fusing a dominantpositive selectable marker with a negative selectable marker. See, e.g.,Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.

Preparation of Cells for Engineering

In some embodiments, preparation of the engineered cells includes one ormore culture and/or preparation steps. The cells for introduction of thenucleic acid encoding the transgenic receptor such as the CAR, may beisolated from a sample, such as a biological sample, e.g., one obtainedfrom or derived from a subject. In some embodiments, the subject fromwhich the cell is isolated is one having the disease or condition or inneed of a cell therapy or to which cell therapy will be administered.The subject in some embodiments is a human in need of a particulartherapeutic intervention, such as the adoptive cell therapy for whichcells are being isolated, processed, and/or engineered.

Accordingly, the cells in some embodiments are primary cells, e.g.,primary human cells. The samples include tissue, fluid, and othersamples taken directly from the subject, as well as samples resultingfrom one or more processing steps, such as separation, centrifugation,genetic engineering (e.g. transduction with viral vector), washing,and/or incubation. The biological sample can be a sample obtaineddirectly from a biological source or a sample that is processed.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples, including processed samples derivedtherefrom.

In some aspects, the sample from which the cells are derived or isolatedis blood or a blood-derived sample, or is or is derived from anapheresis or leukapheresis product. Exemplary samples include wholeblood, peripheral blood mononuclear cells (PBMCs), leukocytes, bonemarrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node,gut associated lymphoid tissue, mucosa associated lymphoid tissue,spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon,kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries,tonsil, or other organ, and/or cells derived therefrom. Samples include,in the context of cell therapy, e.g., adoptive cell therapy, samplesfrom autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T celllines. The cells in some embodiments are obtained from a xenogeneicsource, for example, from mouse, rat, non-human primate, and pig.

In some embodiments, isolation of the cells includes one or morepreparation and/or non-affinity based cell separation steps. In someexamples, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In some examples, cells are separatedbased on one or more property, such as density, adherent properties,size, sensitivity and/or resistance to particular components.

In some examples, cells from the circulating blood of a subject areobtained, e.g., by apheresis or leukapheresis. The samples, in someaspects, contain lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and/or platelets, and in some aspects contains cells other thanred blood cells and platelets.

In some embodiments, the blood cells collected from the subject arewashed, e.g., to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In someembodiments, the cells are washed with phosphate buffered saline (PBS).In some embodiments, the wash solution lacks calcium and/or magnesiumand/or many or all divalent cations. In some aspects, a washing step isaccomplished a semi-automated “flow-through” centrifuge (for example,the Cobe 2991 cell processor, Baxter) according to the manufacturer'sinstructions. In some aspects, a washing step is accomplished bytangential flow filtration (TFF) according to the manufacturer'sinstructions. In some embodiments, the cells are resuspended in avariety of biocompatible buffers after washing, such as, for example,Ca⁺⁺/Mg⁺⁺ free PBS. In certain embodiments, components of a blood cellsample are removed and the cells directly resuspended in culture media.

In some embodiments, the methods include density-based cell separationmethods, such as the preparation of white blood cells from peripheralblood by lysing the red blood cells and centrifugation through a Percollor Ficoll gradient.

In some embodiments, the isolation methods include the separation ofdifferent cell types based on the expression or presence in the cell ofone or more specific molecules, such as surface markers, e.g., surfaceproteins, intracellular markers, or nucleic acid. In some embodiments,any known method for separation based on such markers may be used. Insome embodiments, the separation is affinity- or immunoaffinity-basedseparation. For example, the isolation in some aspects includesseparation of cells and cell populations based on the cells' expressionor expression level of one or more markers, typically cell surfacemarkers, for example, by incubation with an antibody or binding partnerthat specifically binds to such markers, followed generally by washingsteps and separation of cells having bound the antibody or bindingpartner, from those cells having not bound to the antibody or bindingpartner.

Such separation steps can be based on positive selection, in which thecells having bound the reagents are retained for further use, and/ornegative selection, in which the cells having not bound to the antibodyor binding partner are retained. In some examples, both fractions areretained for further use. In some aspects, negative selection can beparticularly useful where no antibody is available that specificallyidentifies a cell type in a heterogeneous population, such thatseparation is best carried out based on markers expressed by cells otherthan the desired population.

The separation need not result in 100% enrichment or removal of aparticular cell population or cells expressing a particular marker. Forexample, positive selection of or enrichment for cells of a particulartype, such as those expressing a marker, refers to increasing the numberor percentage of such cells, but need not result in a complete absenceof cells not expressing the marker. Likewise, negative selection,removal, or depletion of cells of a particular type, such as thoseexpressing a marker, refers to decreasing the number or percentage ofsuch cells, but need not result in a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out,where the positively or negatively selected fraction from one step issubjected to another separation step, such as a subsequent positive ornegative selection. In some examples, a single separation step candeplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types.

For example, in some aspects, specific subpopulations of T cells, suchas cells positive or expressing high levels of one or more surfacemarkers, e.g., CD28⁺, CD62L⁺, CCR7⁺, CD27⁺, CD127⁺, CD4⁺, CD8⁺, CD45RA⁺,and/or CD45RO⁺ T cells, are isolated by positive or negative selectiontechniques.

For example, CD3⁺, CD28⁺ T cells can be positively selected usingCD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 TCell Expander).

In some embodiments, isolation is carried out by enrichment for aparticular cell population by positive selection, or depletion of aparticular cell population, by negative selection. In some embodiments,positive or negative selection is accomplished by incubating cells withone or more antibodies or other binding agent that specifically bind toone or more surface markers expressed or expressed (marker⁺) at arelatively higher level (marker^(high)) on the positively or negativelyselected cells, respectively.

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In some aspects, aCD4⁺ or CD8⁺ selection step is used to separate CD4⁺ helper and CD8⁺cytotoxic T cells. Such CD4⁺ and CD8⁺ populations can be further sortedinto sub-populations by positive or negative selection for markersexpressed or expressed to a relatively higher degree on one or morenaive, memory, and/or effector T cell subpopulations.

In some embodiments, CD8⁺ cells are further enriched for or depleted ofnaive, central memory, effector memory, and/or central memory stemcells, such as by positive or negative selection based on surfaceantigens associated with the respective subpopulation. In someembodiments, enrichment for central memory T (T_(CM)) cells is carriedout to increase efficacy, such as to improve long-term survival,expansion, and/or engraftment following administration, which in someaspects is particularly robust in such sub-populations. See Terakuraetal. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother.35(9):689-701. In some embodiments, combining T_(cm)-enriched CD8⁺ Tcells and CD4⁺ T cells further enhances efficacy.

In embodiments, memory T cells are present in both CD62L⁺ and CD62L⁻subsets of CD8⁺ peripheral blood lymphocytes. PBMC can be enriched foror depleted of CD62L⁻CD8⁺ and/or CD62L⁺CD8⁺ fractions, such as usinganti-CD8 and anti-CD62L antibodies.

In some embodiments, the enrichment for central memory T (T_(CM)) cellsis based on positive or high surface expression of CD45RO, CD62L, CCR7,CD28, CD3, and/or CD 127; in some aspects, it is based on negativeselection for cells expressing or highly expressing CD45RA and/orgranzyme B. In some aspects, isolation of a CD8⁺ population enriched forT_(CM) cells is carried out by depletion of cells expressing CD4, CD14,CD45RA, and positive selection or enrichment for cells expressing CD62L.In one aspect, enrichment for central memory T (T_(CM)) cells is carriedout starting with a negative fraction of cells selected based on CD4expression, which is subjected to a negative selection based onexpression of CD14 and CD45RA, and a positive selection based on CD62L.Such selections in some aspects are carried out simultaneously and inother aspects are carried out sequentially, in either order. In someaspects, the same CD4 expression-based selection step used in preparingthe CD8⁺ cell population or subpopulation, also is used to generate theCD4⁺ cell population or sub-population, such that both the positive andnegative fractions from the CD4-based separation are retained and usedin subsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

In a particular example, a sample of PBMCs or other white blood cellsample is subjected to selection of CD4⁺ cells, where both the negativeand positive fractions are retained. The negative fraction then issubjected to negative selection based on expression of CD14 and CD45RAor CD19, and positive selection based on a marker characteristic ofcentral memory T cells, such as CD62L or CCR7, where the positive andnegative selections are carried out in either order.

CD4⁺ T helper cells are sorted into naïve, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4⁺ lymphocytes can be obtained by standard methods. In someembodiments, naive CD4⁺ T lymphocytes are CD45RO⁻, CD45RA⁺, CD62L⁺, CD4⁺T cells. In some embodiments, central memory CD4⁺ cells are CD62L⁺ andCD45RO⁺. In some embodiments, effector CD4⁺ cells are CD62L⁻ andCD45RO⁻.

In one example, to enrich for CD4⁺ cells by negative selection, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody orbinding partner is bound to a solid support or matrix, such as amagnetic bead or paramagnetic bead, to allow for separation of cells forpositive and/or negative selection. For example, in some embodiments,the cells and cell populations are separated or isolated usingimmunomagnetic (or affinitymagnetic) separation techniques (reviewed inMethods in Molecular Medicine, vol. 58: Metastasis Research Protocols,Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A.Brooks and U. Schumacher © Humana Press Inc., Totowa, N.J.).

In some aspects, the sample or composition of cells to be separated isincubated with small, magnetizable or magnetically responsive material,such as magnetically responsive particles or microparticles, such asparamagnetic beads (e.g., such as Dynalbeads or MACS beads). Themagnetically responsive material, e.g., particle, generally is directlyor indirectly attached to a binding partner, e.g., an antibody, thatspecifically binds to a molecule, e.g., surface marker, present on thecell, cells, or population of cells that it is desired to separate,e.g., that it is desired to negatively or positively select.

In some embodiments, the magnetic particle or bead comprises amagnetically responsive material bound to a specific binding member,such as an antibody or other binding partner. There are many well-knownmagnetically responsive materials used in magnetic separation methods.Suitable magnetic particles include those described in Molday, U.S. Pat.No. 4,452,773, and in European Patent Specification EP 452342 B, whichare hereby incorporated by reference. Colloidal sized particles, such asthose described in Owen U.S. Pat. No. 4,795,698, and Liberti et al.,U.S. Pat. No. 5,200,084 are other examples.

The incubation generally is carried out under conditions whereby theantibodies or binding partners, or molecules, such as secondaryantibodies or other reagents, which specifically bind to such antibodiesor binding partners, which are attached to the magnetic particle orbead, specifically bind to cell surface molecules if present on cellswithin the sample.

In some aspects, the sample is placed in a magnetic field, and thosecells having magnetically responsive or magnetizable particles attachedthereto will be attracted to the magnet and separated from the unlabeledcells. For positive selection, cells that are attracted to the magnetare retained; for negative selection, cells that are not attracted(unlabeled cells) are retained. In some aspects, a combination ofpositive and negative selection is performed during the same selectionstep, where the positive and negative fractions are retained and furtherprocessed or subject to further separation steps.

In certain embodiments, the magnetically responsive particles are coatedin primary antibodies or other binding partners, secondary antibodies,lectins, enzymes, or streptavidin. In certain embodiments, the magneticparticles are attached to cells via a coating of primary antibodiesspecific for one or more markers. In certain embodiments, the cells,rather than the beads, are labeled with a primary antibody or bindingpartner, and then cell-type specific secondary antibody- or otherbinding partner (e.g., streptavidin)-coated magnetic particles, areadded. In certain embodiments, streptavidin-coated magnetic particlesare used in conjunction with biotinylated primary or secondaryantibodies.

In some embodiments, the magnetically responsive particles are leftattached to the cells that are to be subsequently incubated, culturedand/or engineered; in some aspects, the particles are left attached tothe cells for administration to a patient. In some embodiments, themagnetizable or magnetically responsive particles are removed from thecells. Methods for removing magnetizable particles from cells are knownand include, e.g., the use of competing non-labeled antibodies, andmagnetizable particles or antibodies conjugated to cleavable linkers. Insome embodiments, the magnetizable particles are biodegradable.

In some embodiments, the affinity-based selection is viamagnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn,Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable ofhigh-purity selection of cells having magnetized particles attachedthereto. In certain embodiments, MACS operates in a mode wherein thenon-target and target species are sequentially eluted after theapplication of the external magnetic field. That is, the cells attachedto magnetized particles are held in place while the unattached speciesare eluted. Then, after this first elution step is completed, thespecies that were trapped in the magnetic field and were prevented frombeing eluted are freed in some manner such that they can be eluted andrecovered. In certain embodiments, the non-target cells are labelled anddepleted from the heterogeneous population of cells.

In certain embodiments, the isolation or separation is carried out usinga system, device, or apparatus that carries out one or more of theisolation, cell preparation, separation, processing, incubation,culture, and/or formulation steps of the methods. In some aspects, thesystem is used to carry out each of these steps in a closed or sterileenvironment, for example, to minimize error, user handling and/orcontamination. In one example, the system is a system as described inInternational Patent Application, Publication Number WO2009/072003, orUS 20110003380 A1.

In some embodiments, the system or apparatus carries out one or more,e.g., all, of the isolation, processing, engineering, and formulationsteps in an integrated or self-contained system, and/or in an automatedor programmable fashion. In some aspects, the system or apparatusincludes a computer and/or computer program in communication with thesystem or apparatus, which allows a user to program, control, assess theoutcome of, and/or adjust various aspects of the processing, isolation,engineering, and formulation steps.

In some aspects, the separation and/or other steps is carried out usingCliniMACS system (Miltenyi Biotic), for example, for automatedseparation of cells on a clinical-scale level in a closed and sterilesystem. Components can include an integrated microcomputer, magneticseparation unit, peristaltic pump, and various pinch valves. Theintegrated computer in some aspects controls all components of theinstrument and directs the system to perform repeated procedures in astandardized sequence. The magnetic separation unit in some aspectsincludes a movable permanent magnet and a holder for the selectioncolumn. The peristaltic pump controls the flow rate throughout thetubing set and, together with the pinch valves, ensures the controlledflow of buffer through the system and continual suspension of cells.

The CliniMACS system in some aspects uses antibody-coupled magnetizableparticles that are supplied in a sterile, non-pyrogenic solution. Insome embodiments, after labelling of cells with magnetic particles thecells are washed to remove excess particles. A cell preparation bag isthen connected to the tubing set, which in turn is connected to a bagcontaining buffer and a cell collection bag. The tubing set consists ofpre-assembled sterile tubing, including a pre-column and a separationcolumn, and are for single use only. After initiation of the separationprogram, the system automatically applies the cell sample onto theseparation column. Labelled cells are retained within the column, whileunlabeled cells are removed by a series of washing steps. In someembodiments, the cell populations for use with the methods describedherein are unlabeled and are not retained in the column. In someembodiments, the cell populations for use with the methods describedherein are labeled and are retained in the column. In some embodiments,the cell populations for use with the methods described herein areeluted from the column after removal of the magnetic field, and arecollected within the cell collection bag.

In certain embodiments, separation and/or other steps are carried outusing the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACSProdigy system in some aspects is equipped with a cell processing unitythat permits automated washing and fractionation of cells bycentrifugation. The CliniMACS Prodigy system can also include an onboardcamera and image recognition software that determines the optimal cellfractionation endpoint by discerning the macroscopic layers of thesource cell product. For example, peripheral blood is automaticallyseparated into erythrocytes, white blood cells and plasma layers. TheCliniMACS Prodigy system can also include an integrated cell cultivationchamber which accomplishes cell culture protocols such as, e.g., celldifferentiation and expansion, antigen loading, and long-term cellculture. Input ports can allow for the sterile removal and replenishmentof media and cells can be monitored using an integrated microscope. See,e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakuraetal. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother.35(9):689-701.

In some embodiments, a cell population described herein is collected andenriched (or depleted) via flow cytometry, in which cells stained formultiple cell surface markers are carried in a fluidic stream. In someembodiments, a cell population described herein is collected andenriched (or depleted) via preparative scale (FACS)-sorting. In certainembodiments, a cell population described herein is collected andenriched (or depleted) by use of microelectromechanical systems (MEMS)chips in combination with a FACS-based detection system (see, e.g., WO2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al.(2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeledwith multiple markers, allowing for the isolation of well-defined T cellsubsets at high purity.

In some embodiments, the antibodies or binding partners are labeled withone or more detectable marker, to facilitate separation for positiveand/or negative selection. For example, separation may be based onbinding to fluorescently labeled antibodies. In some examples,separation of cells based on binding of antibodies or other bindingpartners specific for one or more cell surface markers are carried in afluidic stream, such as by fluorescence-activated cell sorting (FACS),including preparative scale (FACS) and/or microelectromechanical systems(MEMS) chips, e.g., in combination with a flow-cytometric detectionsystem. Such methods allow for positive and negative selection based onmultiple markers simultaneously.

In some embodiments, the preparation methods include steps for freezing,e.g., cryopreserving, the cells, either before or after isolation,incubation, and/or engineering. In some embodiments, the freeze andsubsequent thaw step removes granulocytes and, to some extent, monocytesin the cell population. In some embodiments, the cells are suspended ina freezing solution, e.g., following a washing step to remove plasma andplatelets. Any of a variety of known freezing solutions and parametersin some aspects may be used. One example involves using PBS containing20% DMSO and 8% human serum albumin (HSA), or other suitable cellfreezing media. This is then diluted 1:1 with media so that the finalconcentration of DMSO and HSA are 10% and 4%, respectively. The cellsare generally then frozen to −80° C. at a rate of 1° per minute andstored in the vapor phase of a liquid nitrogen storage tank.

In some embodiments, the provided methods include cultivation,incubation, culture, and/or genetic engineering steps. For example, insome embodiments, provided are methods for incubating and/or engineeringthe depleted cell populations and culture-initiating compositions.

Thus, in some embodiments, the cell populations are incubated in aculture-initiating composition. The incubation and/or engineering may becarried out in a culture vessel, such as a unit, chamber, well, column,tube, tubing set, valve, vial, culture dish, bag, or other container forculture or cultivating cells.

In some embodiments, the cells are incubated and/or cultured prior to orin connection with genetic engineering. The incubation steps can includeculture, cultivation, stimulation, activation, and/or propagation. Insome embodiments, the compositions or cells are incubated in thepresence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a recombinant antigen receptor.

The conditions can include one or more of particular media, temperature,oxygen content, carbon dioxide content, time, agents, e.g., nutrients,amino acids, antibiotics, ions, and/or stimulatory factors, such ascytokines, chemokines, antigens, binding partners, fusion proteins,recombinant soluble receptors, and any other agents designed to activatethe cells.

In some embodiments, the stimulating conditions or agents include one ormore agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In some aspects, theagent turns on or initiates TCR/CD3 intracellular signaling cascade in aT cell. Such agents can include antibodies, such as those specific for aTCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28,for example, bound to solid support such as a bead, and/or one or morecytokines. Optionally, the expansion method may further comprise thestep of adding anti-CD3 and/or anti CD28 antibody to the culture medium(e.g., at a concentration of at least about 0.5 ng/ml). In someembodiments, the stimulating agents include IL-2 and/or IL-15, forexample, an IL-2 concentration of at least about 10 units/mL.

In some aspects, incubation is carried out in accordance with techniquessuch as those described in U.S. Pat. No. 6,040,177 to Riddell et al.,Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakuraet al.(2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother.35(9):689-701.

In some embodiments, the T cells are expanded by adding to theculture-initiating composition feeder cells, such as non-dividingperipheral blood mononuclear cells (PBMC), (e.g., such that theresulting population of cells contains at least about 5, 10, 20, or 40or more PBMC feeder cells for each T lymphocyte in the initialpopulation to be expanded); and incubating the culture (e.g. for a timesufficient to expand the numbers of T cells). In some aspects, thenon-dividing feeder cells can comprise gamma-irradiated PBMC feedercells. In some embodiments, the PBMC are irradiated with gamma rays inthe range of about 3000 to 3600 rads to prevent cell division. In someaspects, the feeder cells are added to culture medium prior to theaddition of the populations of T cells.

In some embodiments, the stimulating conditions include temperaturesuitable for the growth of human T lymphocytes, for example, at leastabout 25 degrees Celsius, generally at least about 30 degrees, andgenerally at or about 37 degrees Celsius. Optionally, the incubation mayfurther comprise adding non-dividing EBV-transformed lymphoblastoidcells (LCL) as feeder cells. LCL can be irradiated with gamma rays inthe range of about 6000 to 10,000 rads. The LCL feeder cells in someaspects is provided in any suitable amount, such as a ratio of LCLfeeder cells to initial T lymphocytes of at least about 10:1.

In embodiments, antigen-specific T cells, such as antigen-specific CD4+and/or CD8+ T cells, are obtained by stimulating naive or antigenspecific T lymphocytes with antigen. For example, antigen-specific Tcell lines or clones can be generated to cytomegalovirus antigens byisolating T cells from infected subjects and stimulating the cells invitro with the same antigen.

X. Compositions and Formulations

Also provided are compositions including the cells for administration,including pharmaceutical compositions and formulations, such as unitdose form compositions including the number of cells for administrationin a given dose or fraction thereof. The pharmaceutical compositions andformulations generally include one or more optional pharmaceuticallyacceptable carrier or excipient. In some embodiments, the compositionincludes at least one additional therapeutic agent.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

In some aspects, the choice of carrier is determined in part by theparticular cell and/or by the method of administration. Accordingly,there are a variety of suitable formulations. For example, thepharmaceutical composition can contain preservatives. Suitablepreservatives may include, for example, methylparaben, propylparaben,sodium benzoate, and benzalkonium chloride. In some aspects, a mixtureof two or more preservatives is used. The preservative or mixturesthereof are typically present in an amount of about 0.0001% to about 2%by weight of the total composition. Carriers are described, e.g., byRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG).

Buffering agents in some aspects are included in the compositions.Suitable buffering agents include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. In some aspects, a mixture of two or more buffering agents isused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition. Methods for preparing administrable pharmaceuticalcompositions are known. Exemplary methods are described in more detailin, for example, Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulations can include aqueous solutions. The formulation orcomposition may also contain more than one active ingredient useful forthe particular indication, disease, or condition being treated with thecells, preferably those with activities complementary to the cells,where the respective activities do not adversely affect one another.Such active ingredients are suitably present in combination in amountsthat are effective for the purpose intended. Thus, in some embodiments,the pharmaceutical composition further includes other pharmaceuticallyactive agents or drugs, such as chemotherapeutic agents, e.g.,asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,paclitaxel, rituximab, vinblastine, and/or vincristine.

The pharmaceutical composition in some embodiments contains the cells inamounts effective to treat or prevent the disease or condition, such asa therapeutically effective or prophylactically effective amount.Therapeutic or prophylactic efficacy in some embodiments is monitored byperiodic assessment of treated subjects. The desired dosage can bedelivered by a single bolus administration of the cells, by multiplebolus administrations of the cells, or by continuous infusionadministration of the cells.

In some embodiments, the composition includes the cells in an amounteffective to reduce burden of the disease or condition, and/or in anamount that does not result in CRS or severe CRS in the subject and/orto effect any of the other outcomes of the methods as described herein.

The cells and compositions may be administered using standardadministration techniques, formulations, and/or devices. Administrationof the cells can be autologous or heterologous. For example,immunoresponsive cells or progenitors can be obtained from one subject,and administered to the same subject or a different, compatible subject.Peripheral blood derived immunoresponsive cells or their progeny (e.g.,in vivo, ex vivo or in vitro derived) can be administered via localizedinjection, including catheter administration, systemic injection,localized injection, intravenous injection, or parenteraladministration. When administering a therapeutic composition (e.g., apharmaceutical composition containing a genetically modifiedimmunoresponsive cell), it will generally be formulated in a unit dosageinjectable form (solution, suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual, or suppository administration. In some embodiments, the cellpopulations are administered parenterally. The term “parenteral,” asused herein, includes intravenous, intramuscular, subcutaneous, rectal,vaginal, and intraperitoneal administration. In some embodiments, thecells are administered to the subject using peripheral systemic deliveryby intravenous, intraperitoneal, or subcutaneous injection.

Compositions in some embodiments are provided as sterile liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,dispersions, or viscous compositions, which may in some aspects bebuffered to a selected pH. Liquid preparations are normally easier toprepare than gels, other viscous compositions, and solid compositions.Additionally, liquid compositions are somewhat more convenient toadminister, especially by injection. Viscous compositions, on the otherhand, can be formulated within the appropriate viscosity range toprovide longer contact periods with specific tissues. Liquid or viscouscompositions can comprise carriers, which can be a solvent or dispersingmedium containing, for example, water, saline, phosphate bufferedsaline, polyoi (for example, glycerol, propylene glycol, liquidpolyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cellsin a solvent, such as in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose,dextrose, or the like. The compositions can contain auxiliary substancessuch as wetting, dispersing, or emulsifying agents (e.g.,methylcellulose), pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, and/or colors, dependingupon the route of administration and the preparation desired. Standardtexts may in some aspects be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of the injectable pharmaceutical form can bebrought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

XII. Articles of Manufacture

Also provided are articles of manufacture, such as kits and devices, forthe administration of the cells to subjects in according to the providedmethods for adoptive cell therapy, and for storage and administration ofthe cells and compositions.

The articles of manufacture include one or more containers, typically aplurality of containers, packaging material, and a label or packageinsert on or associated with the container or containers and/orpackaging, generally including instructions for administration of thecells to a subject.

The containers generally contain the cells to be administered, e.g., oneor more unit doses thereof. The article of manufacture typicallyincludes a plurality of containers, each containing a single unit doseof the cells. The unit dose may be an amount or number of the cells tobe administered to the subject in the first dose or twice the number (ormore) the cells to be administered in the first or consecutive dose(s).It may be the lowest dose or lowest possible dose of the cells thatwould be administered to the subject in connection with theadministration method. In some embodiments, the unit dose is the minimumnumber of cells or number of cells that would be administered in asingle dose to any subject having a particular disease or condition orany subject, according to the methods herein. For example, the unit dosein some aspects may include a minimum number of cells that would beadministered to a patient of a relatively lower body weight and/or withrelatively low disease burden, such that one and in some cases more thanone unit dose is administered to a given subject as a first dose and oneor more than one unit dose is administered to a given subject in one ormore consecutive dose, e.g., according to the provided methods. In someembodiments, the number of cells in the unit dose is the number of cellsor number of recombinant receptor-expressing or CAR-expressing cellsthat it is desired to administer to a particular subject in a firstdose, such as a subject from which the cells have been derived. In someembodiments, the cells have been derived from the subject to be treatedby methods as provided herein or in need thereof.

In some embodiments, each of the containers individually comprises aunit dose of the cells, e.g., including the same or substantially thesame number of cells. Thus in some embodiments, each of the containerscomprises the same or approximately or substantially the same number ofcells or number of recombinant receptor-expressing cells. In someembodiments, the unit dose includes less than about 1×10⁸, less thanabout 5×10⁷, less than about 1×10⁶ or less than about 5×10⁵ of theengineered cells, of total cells, of T cells, or PBMCs, per kg of thesubject to be treated and/or from which the cells have been derived. Insome embodiments, each unit dose contains at or about 2×10⁶, 5×10⁶,1×10⁷, 5×10⁷, or 1×10⁸ engineered cells, total cells, T cells, or PBMCs.

Suitable containers include, for example, bottles, vials, syringes, andflexible bags, such as infusion bags. In particular embodiments, thecontainers are bags, e.g., flexible bags, such as those suitable forinfusion of cells to subjects, e.g., flexible plastic or PVC bags,and/or IV solution bags. The bags in some embodiments are sealableand/or able to be sterilized, so as to provide sterile solution anddelivery of the cells and compositions. In some embodiments, thecontainers, e.g., bags, have a capacity of at or about or at least at orabout 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or1000 ml capacity, such as between at or about 10 and at or about 100 orbetween at or about 10 and at or about 500 mL capacity. In someembodiments, the containers, e.g., bags, are and/or are made frommaterial which is stable and/or provide stable storage and/ormaintenance of cells at one or more of various temperatures, such as incold temperatures, e.g. below at or about or at or about −20° C., −80°C., −120° C., 135° C. and/or temperatures suitable for cryopreservation,and/or other temperatures, such as temperatures suitable for thawing thecells and body temperature such as at or about 37° C., for example, topermit thawing, e.g., at the subject's location or location oftreatment, e.g., at bedside, immediately prior to treatment.

The containers may be formed from a variety of materials such as glassor plastic. In some embodiments, the container has one or more port,e.g., sterile access ports, for example, for connection of tubing orcannulation to one or more tubes, e.g., for intravenous or otherinfusion and/or for connection for purposes of transfer to and fromother containers, such as cell culture and/or storage bags or othercontainers. Exemplary containers include infusion bags, intravenoussolution bags, vials, including those with stoppers pierceable by aneedle for injection.

The article of manufacture may further include a package insert or labelwith one or more pieces of identifying information and/or instructionsfor use. In some embodiments, the information or instructions indicatesthat the contents can or should be used to treat a particular conditionor disease, and/or providing instructions therefor. The label or packageinsert may indicate that the contents of the article of manufacture areto be used for treating the disease or condition. In some embodiments,the label or package insert provides instructions to treat a subject,e.g., the subject from which the cells have been derived, via a methodinvolving the administration of a first and one or more consecutivedoses of the cells, e.g., according to any of the embodiments of theprovided methods. In some embodiments, the instructions specifyadministration, in a first dose, of one unit dose, e.g., the contents ofa single individual container in the article of manufacture, followed byone or more consecutive doses at a specified time point or within aspecified time window and/or after the detection of the presence orabsence or amount or degree of one or more factors or outcomes in thesubject.

In some embodiments, the instructions specify administering a pluralityof the unit doses to the subject by carrying out a first administrationand a consecutive administration. In some embodiments, the firstadministration comprises delivering one of said unit doses to thesubject and the consecutive administration comprises administering oneor a plurality of said unit doses to the subject.

In some embodiments, the instructions specify that the consecutiveadministration is to be carried out at a time between about 15 and about27 days or between about 9 and about 35 days, e.g., at or about 21 days,following the first administration, e.g., following the initiation ofthe first administration or the prior administration. In someembodiments, the instructions specify that the consecutive dose is to beadministered at a time after which it has been determined that a serumlevel of a factor indicative of cytokine-release syndrome (CRS) in thesubject is less than about 10 times, less than about 25 times, and/orless than about 50 times the serum level of the indicator in the subjectimmediately prior to said first administration, and/or that an indicatorof CRS has peaked and is declining, and/or that the subject does notexhibit a detectable adaptive host immune response specific for thereceptor, e.g., CAR, expressed by the cells.

In some embodiments, the label or package insert or packaging comprisesan identifier to indicate the specific identity of the subject fromwhich the cells are derived and/or are to be administered. In the caseof autologous transfer, the identity of the subject from which the cellsare derived is the same as the identity of the subject to which thecells are to be administered. Thus, the identifying information mayspecify that the cells are to be administered to a particular patient,such as the one from which the cells were originally derived. Suchinformation may be present in the packaging material and/or label in theform of a bar code or other coded identifier, or may indication the nameand/or other identifying characteristics of the subject.

The article of manufacture in some embodiments includes one or more,typically a plurality, of containers containing compositions comprisingthe cells, e.g., individual unit dose forms thereof, and further includeone or more additional containers with a composition contained thereinwhich includes a further agent, such as a cytotoxic or otherwisetherapeutic agent, for example, which is to be administered incombination, e.g., simultaneously or sequentially in any order, with thecells. Alternatively, or additionally, the article of manufacture mayfurther include another or the same container comprising apharmaceutically-acceptable buffer. It may further include othermaterials such as other buffers, diluents, filters, tubing, needles,and/or syringes.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,“a” or “an” means “at least one” or “one or more.” It is understood thataspects and variations described herein include “consisting” and/or“consisting essentially of” aspects and variations.

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein, a composition refers to any mixture of two or moreproducts, substances, or compounds, including cells. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, a statement that a cell or population of cells is“positive” for a particular marker refers to the detectable presence onor in the cell of a particular marker, typically a surface marker. Whenreferring to a surface marker, the term refers to the presence ofsurface expression as detected by flow cytometry, for example, bystaining with an antibody that specifically binds to the marker anddetecting said antibody, wherein the staining is detectable by flowcytometry at a level substantially above the staining detected carryingout the same procedure with an isotype-matched control under otherwiseidentical conditions and/or at a level substantially similar to that forcell known to be positive for the marker, and/or at a levelsubstantially higher than that for a cell known to be negative for themarker.

As used herein, a statement that a cell or population of cells is“negative” for a particular marker refers to the absence of substantialdetectable presence on or in the cell of a particular marker, typicallya surface marker. When referring to a surface marker, the term refers tothe absence of surface expression as detected by flow cytometry, forexample, by staining with an antibody that specifically binds to themarker and detecting said antibody, wherein the staining is not detectedby flow cytometry at a level substantially above the staining detectedcarrying out the same procedure with an isotype-matched control underotherwise identical conditions, and/or at a level substantially lowerthan that for cell known to be positive for the marker, and/or at alevel substantially similar as compared to that for a cell known to benegative for the marker.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

All publications, including patent documents, scientific articles anddatabases, referred to in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication were individually incorporated by reference. If adefinition set forth herein is contrary to or otherwise inconsistentwith a definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

XIII. Exemplary Embodiments

Among the embodiments provided herein are:

1. A method of treatment, comprising:

-   -   (a) administering to a subject having a disease or condition a        first dose of cells expressing a chimeric antigen receptor        (CAR), said first dose comprising no more than about 1×10⁶ of        the cells per kilogram body weight of the subject, no more than        about 1×10⁸ of the cells, and/or no more than about 1×10⁸ of the        cells/m² of the subject; and    -   (b) administering to the subject a consecutive dose of cells        expressing a CAR at a time point that is at least or more than        about 14 days after and less than about 28 days after initiation        of said administration in (a).

2. The method of embodiment 1, wherein, at the time of theadministration in (b):

-   -   (i) the serum level in the subject of a factor indicative of        cytokine release syndrome (CRS) is less than about 10 times,        less than about 25 times, and/or less than about 50 times that        in the subject immediately prior to said administration in (a);        and/or    -   (ii) the subject does not exhibit grade 3 or higher        neurotoxicity; and/or    -   (iii) a CRS-related outcome or symptom of neurotoxicity in the        subject following said administration of said first dose has        reached a peak level and begun to decline following the        administration in (a); and/or    -   (iv) the subject does not exhibit a detectable humoral or        cell-mediated immune response against the CAR expressed by the        cells of said first dose.

3. A method of treatment, comprising:

-   -   (a) administering to a subject a first dose of cells expressing        a chimeric antigen receptor (CAR), said first dose comprising        the cells in an amount sufficient to reduce burden of a disease        or condition in the subject; and    -   (b) administering to the subject a consecutive dose of        CAR-expressing cells at a time at which:    -   (i) a clinical risk for neurotoxicity, cytokine-release syndrome        (CRS), macrophage activation syndrome, or tumor lysis syndrome,        is not present or has passed or has subsided following said        administration in (a), (ii) a biochemical readout evidencing        (CRS), neurotoxicity, macrophage activation syndrome, or tumor        lysis syndrome, is not present or has passed or has subsided        following said administration in (a), and/or (iii) a serum level        of a factor indicative of cytokine-release syndrome (CRS) or        neurotoxicity in the subject is less than about 10 times, less        than about 25 times, and/or less than about 50 times the serum        level of said indicator in the subject immediately prior to said        administration in (a); and    -   the subject does not exhibit a detectable adaptive host immune        response specific for the CAR expressed by the cells of said        first dose.

4. A method of treatment, comprising:

-   -   (a) administering to a subject a first dose of cells expressing        a chimeric antigen receptor (CAR), said first dose comprising        the cells in an amount sufficient to reduce burden of a disease        or condition in the subject; and    -   (b) administering to the subject a consecutive dose of        CAR-expressing cells, at a time after a neurotoxicity and/or        CRS-related outcome in the subject has reached a peak level and        begun to decline following said administration in (a) and at        which the subject does not exhibit a detectable humoral or        cell-mediated immune response against the CAR expressed by the        cells of said first dose.

5. The method of embodiment 3 or embodiment 4, wherein:

-   -   (i) the administration in (a) does not induce severe CRS in the        subject or does not induce CRS in the subject;    -   (ii) the administration in (a) does not induce grade 3 or higher        neurotoxicity in the subject;    -   (iii) based on clinical data, the administration of the dose of        cells in (a) does not induce severe CRS in a majority of        subjects so-treated; and/or    -   (iv) based on clinical data, the administration of the dose of        cells in (a) does not induce grade 3 or higher neurotoxicity in        a majority of subjects so-treated.

6. The method of any of embodiments 3-5, wherein:

-   -   the time between the initiation of the administration in (a) and        the initiation of the administration in (b) is between about 9        and about 35 days, between about 14 and about 28 days, between        15 and 27 days or is between 17 days and about 21 days, each        inclusive; and/or    -   said first dose comprises no more than about 1×10⁶ of the cells        per kilogram body weight of the subject, no more than about        1×10⁸ of the cells, or no more than about 1×10⁸ of the cells/m²        of the subject.

7. The method of any of embodiments 2-6, wherein the CRS-related outcomeis selected from the group consisting of fever, hypotension, hypoxia,neurologic disturbances, or a serum level of an inflammatory cytokine orC reactive protein (CRP).

8. The method of any of embodiments 2-6, wherein the factor indicativeof CRS is an inflammatory cytokine selected from the group consisting ofinterferon gamma (IFNγ), granulocyte-macrophage colony-stimulatingfactor (GM-CSF), tumor necrosis factor alpha (TNFα), IL-6, IL-10, IL-1β,IL-8, IL-2, MIP-1, Flt-3L, fracktalkine, and IL-5 or is CRP.

9. The method of any of embodiments 2-8, wherein, at the time of theadministration in (b):

-   -   said level of said CRS-related outcome is no more than 50% of        the peak level, is no more than 20% of the peak level, or is no        more than 5% of the peak level, or is at or about the level        immediately prior to the administration in (a); or said serum        level of said factor indicative of CRS is no more than ten times        the level immediately prior to the administration in (a).

10. The method of any of embodiments 2-9, wherein symptoms associatedwith a clinical risk of neurotoxicity and/or grade 3 or higherneurotoxicity are selected from among confusion, delirium, expressiveaphasia, obtundation, myoclonus, lethargy, altered mental status,convulsions, seizure-like activity, seizures (optionally as confirmed byelectroencephalogram [EEG]), elevated levels of beta amyloid (Aβ),elevated levels of glutamate, and elevated levels of oxygen radicals.

11. The method of any of embodiments 1-10, wherein said subject has notreceived a dose of cells expressing the CAR expressed by the cells inthe first dose prior to the administration in (a).

12. The method of any of embodiments 1-11, wherein the CAR expressed bythe cells in the consecutive dose contains at least one immunoreactiveepitope present in the CAR expressed by the cells in the first dose.

13. The method of embodiment 12, wherein the CAR expressed by the cellsin the consecutive dose is identical to the CAR expressed by the cellsin the first dose or is substantially identical to the CAR expressed bythe cells in the first dose.

14. The method of any of embodiments 1-13, wherein the CAR expressed bythe cells in the first dose specifically binds to an antigen expressedby a cell or tissue of the disease or condition or associated with thedisease or condition.

15. The method of embodiment 14, wherein the disease or condition is atumor or a cancer.

16. The method of embodiment 15, wherein the administration in (a) leadsto a reduction in burden of the disease or condition in the subject, asindicated by a reduction in one or more factors indicative of diseaseburden following said administration in (a).

17. The method of embodiment 16, wherein at the time of theadministration in (b), the subject has not relapsed and/or the one ormore factors indicative of disease burden have not increased followingsaid reduction.

18. The method of any of embodiments 1-17, wherein the consecutive doseof cells comprises cells in an amount sufficient for reduction in burdenof a disease or condition in the subject.

19. The method of any of embodiments 16-18, wherein the administrationin (b) leads to a further reduction in burden of the disease orcondition in the subject.

20. The method of any of embodiments 1-19, wherein said administrationof said consecutive dose leads to a reduction in burden of the diseaseor condition in the subject as compared with immediately prior toinitiation of the administration of the consecutive dose.

21. The method of any of embodiments 1-20, wherein the method reducesburden of the disease or condition to a greater degree and/or for agreater period of time as compared to a method comprising an alternativedosing regimen wherein the subject is administered the cells in (a) andthe cells in (b) in a single dose.

22. The method of any of embodiments 16-22, wherein:

-   -   said reduction in burden and/or further reduction in burden        comprises a reduction in total number of cells of the disease in        the subject, in an organ of the subject, in a tissue of the        subject, or in a bodily fluid of the subject, a reduction in        mass or volume of a tumor, and/or a reduction in number and/or        extent of metastases.

23. The method of any of embodiments 1-22, wherein:

-   -   the disease is a cancer and the subject does not exhibit        morphologic disease at the time of initiation of the        administration in (b); and/or    -   the disease is a leukemia or lymphoma and the subject does not        exhibit greater than 5% blast cells in the bone marrow at the        time of the administration in (b).

24. The method of any of embodiments 1-23, wherein the disease orcondition persists following the administration of said first doseand/or the administration of the first dose is not sufficient toeradicate the disease or condition in the subject.

25. The method of any of embodiments 1-24, wherein the subject exhibitsdetectable molecular disease and/or minimum residual disease at the timeof the administration in (b).

26. The method of any of embodiments 1-25, wherein (i) the maximumnumber of CAR-expressing cells, (ii) the area under the curve (AUC) ofCAR-expressing cells over time, and/or (iii) the duration of detectableCAR-expressing cells in the subject following said administration in (b)is greater, as compared to that achieved via a method comprising analternative dosing regimen wherein the subject is administered the cellsin (a) and the cells in (b) as a single dose.

27. The method of any of embodiments 1-26, wherein:

-   -   the method results in a maximum concentration or number of        CAR-expressing cells in the blood of the subject of at least at        or about 10 CAR-expressing cells per microliter, at least 50% of        the total number of peripheral blood mononuclear cells (PBMCs),        at least at least about 1×10⁵ CAR-expressing cells, or at least        5,000 copies of CAR-encoding DNA per micrograms DNA; and/or    -   at day 90 following the initiation of the administration in (a),        CAR-expressing cells are detectable in the blood or serum of the        subject; and/or    -   at day 90 following the initiation of the administration in (a),        the blood of the subject contains at least 20% CAR-expressing        cells, at least 10 CAR-expressing cells per microliter or at        least 1×10⁴ CAR-expressing cells.

28. The method of any of embodiments 1-27, wherein the area under thecurve (AUC) for blood concentration of CAR-expressing cells over timefollowing the administration in (a) is greater as compared to thatachieved via a method comprising an alternative dosing regimen whereinthe subject is administered the cells in (a) and the cells in (b) as asingle dose.

29. The method of any of embodiments 1-28, wherein a CRS-related outcomein the subject at day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14following the administration in (b) is not detectable or is reduced ascompared to a method comprising an alternative dosing regimen whereinthe subject is administered the cells in (b) without having beenadministered the first dose.

30. The method of any of embodiments 1-29, wherein the area under thecurve (AUC) for a serum level of a factor indicative of CRS over time inthe subject following the administration in (b) is lower as compared tothat of a method comprising an alternative dosing regimen wherein thesubject is administered the cells in (b) without having beenadministered the first dose.

31. The method of any of embodiments 15-30, wherein the subject has beentreated with a therapeutic agent targeting the tumor or cancer prior tosaid administration in (a) and is refractory or non-responsive to saidtherapeutic agent at the time of the administration in (a).

32. The method of any of embodiments 1-31, further comprising,subsequent to administration in (a) and before said administration in(b), or prior to administration in (a), assessing a serum level of afactor indicative of CRS, a factor indicative of neurotoxicity, a factorindicative of disease burden, and/or an indicator of a host anti-CARimmune response in said subject.

33. The method of embodiment 32, wherein the factor indicative ofdisease burden is measured and comprises a total number of cells of thedisease in the subject, in an organ of the subject, in a tissue of thesubject, or in a bodily fluid of the subject, molecular detection byflow cytometry or quantitative PCR, mass or volume of a solid tumor, ornumber or extent of metastases.

34. The method of embodiment 32 or embodiment 33, comprising:

-   -   i) assessing a factor indicative of disease burden prior to        administration in (b); and    -   ii) based on the result of the assessment, determining the        consecutive dose of cells to be administered to the subject,        and:    -   iii) if the assessment determines that the subject has        morphologic disease, administering to the subject a consecutive        dose comprising less than or about the same number of        CAR-expressing cells as the number of CAR-expressing cells in        the first dose; and/or if the assessment determines that the        subject has minimal residual disease, administering to the        subject a consecutive dose comprising an increased number of        CAR-expressing cells as compared to the first dose.

35. The method of any of embodiments 1-33, wherein the consecutive dosecomprises about the same number of CAR-expressing cells as the number ofCAR-expressing cells in the first dose.

36. The method of any of embodiments 1-33, wherein the consecutive dosecomprises an increased number of CAR-expressing cells as compared to thefirst dose.

37. A method of treatment, comprising administering a consecutive doseof cells expressing a chimeric antigen receptor (CAR) to a subjectpreviously administered a first dose of cells expressing a CAR, wherein:

-   -   the consecutive dose of cells is administered at a time point        that is at least or more than about 14 days after and less than        about 28 days after initiation of the first dose; and/or    -   the number of CAR-expressing cells administered in the        consecutive dose is increased as compared to the first dose.

38. The method of any of embodiments 1-37, wherein the number of cellsadministered in the first dose is between about 0.5×10⁶ cells/kg bodyweight of the subject and 3×10⁶ cells/kg, between about 0.75×10⁶cells/kg and 2.5×10⁶ cells/kg or between about 1×10⁶ cells/kg and 2×10⁶cells/kg, each inclusive.

39. The method of embodiment 34, 36 or 37, wherein the increased numberis at least 2-fold, 5-fold, or 10-fold greater than the number in thefirst dose.

40. The method of any of embodiments 1-39, wherein the number of cellsadministered in the consecutive dose of CAR-expressing cells comprisesbetween about 2×10⁶ cells per kilogram (cells/kg) body weight and about6×10⁶ cells/kg, between about 2.5×10⁶ cells/kg and about 5.0×10⁶cells/kg, or between about 3.0×10⁶ cells/kg and about 4.0×10⁶ cells/kg,each inclusive.

41. The method of any of embodiments 1-40, wherein the subject does notexhibit cytokine release syndrome (CRS), does not exhibit severe CRS,does not exhibit neurotoxicity, does not exhibit severe neurotoxicity,or does not exhibit neurotoxicity above grade 3 following administrationof the first dose and/or following the administration in of theconsecutive dose.

42. The method of any of embodiments 1-41, wherein the CAR-expressingcells in the first dose expand in the subject following administrationof the first dose and/or following the administration of the consecutivedose.

43. The method of embodiment 42, wherein the expansion is evidenced by(i) an increase in serum CRP level following the administration of thefirst dose and/or consecutive dose as compared to just prior to theadministration, and/or (ii) an increase in level of CAR-encoding nucleicacid in the serum, as measured by qPCR, following the administration ofthe first dose and/or consecutive dose as compared to just prior to theadministration, wherein said increase is optionally at least 1, 2, or3-fold.

44. The method of any of embodiments 1-43, wherein the time between thefirst and consecutive dose is between 15 and 27 days.

45. The method of embodiment 44, wherein the time between the first andconsecutive dose is about 21 days.

46. The method of embodiment 44, wherein the time between the first andconsecutive dose is about 17 days.

47. The method of any of embodiments 1-46, wherein the cells of thefirst dose are administered in a single pharmaceutical compositioncomprising the cells of the first dose and/or wherein the cells of theconsecutive dose are administered in a single pharmaceutical compositioncomprising the cells of the consecutive dose.

48. The method of any of embodiments 1-47, wherein:

-   -   the first dose is a split dose, wherein the cells of the first        dose are administered in a plurality of compositions,        collectively comprising the cells of the first dose, over a        period of no more than three days; and/or    -   the consecutive dose is a split dose, wherein the cells of the        consecutive dose are administered in a plurality of        compositions, collectively comprising the cells of the        consecutive dose, over a period of no more than three days.

49. The method of any of embodiments 1-48, wherein the method furthercomprises administering a chemotherapeutic agent prior to theadministration in (a) and/or the administration in (b) or wherein thesubject has been previously treated with a chemotherapeutic agent priorto the administration of the first dose.

50. The method of embodiment 49, wherein the chemotherapeutic agentcomprises an agent selected from the group consisting ofcyclophosphamide, fludarabine, and/or a combination thereof.

51. The method of embodiment 49 or 50, wherein the administration of thechemotherapeutic agent comprises administration of a chemotherapeuticagent prior to the administration in (a) and optionally not prior to theadministration in (b).

52. The method of any of embodiments 49-51, wherein the chemotherapeuticagent is administered between 2 and 5 days prior to the administrationin (a) and/or is administered between 2 and 5 days prior to theadministration in (b).

53. The method of any of embodiments 49-52, wherein the chemotherapeuticagent is administered at a dose of between at or about 1 g/m² of thesubject and at or about 100 g/m², 15 g/m² of the subject and 50 g/m² ofthe subject, 0.5 g/m² of the subject and 5 g/m² of the subject, on g/m²of the subject and at or about 3 g/m² of the subject.

54. The method of any of embodiments 1-53, wherein the subject hasreceived cryoreductive chemotherapy prior to the administration or thefirst dose or the method further comprises the administration ofcryoreductive chemotherapy prior to the administration of the firstdose.

55. The method of any of embodiments 49-54, wherein the chemotherapeuticagent comprises conditioning chemotherapy, which reduces burden of thedisease or condition in the subject.

56. A method of providing consolidating treatment, comprisingadministering to a subject a consecutive dose of cells expressing achimeric antigen receptor (CAR), wherein:

-   -   prior to said administration, the subject has received a        previous dose of CAR-expressing in an amount sufficient to        reduce burden of a disease or condition in the subject; and    -   at the time of administration, a serum level of a factor        indicative of cytokine-release syndrome (CRS) in the subject is        less than about 10 times, less than about 25 times, and/or less        than about 50 times the serum level of said indicator in the        subject immediately prior to said previous dose and the subject        does not exhibit a detectable adaptive host immune response        specific for the CAR expressed by the cells of said previous        dose; and/or the time between said previous and consecutive        doses is greater than about 14 days and less than about 28 days.

57. The method of embodiment 56, wherein the number of cells in theconsecutive dose of CAR-expressing cells comprises between about 2×10⁶cells per kilogram (cells/kg) body weight and about 6×10⁶ cells/kg,between about 2.5×10⁶ cells/kg and about 5.0×10⁶ cells/kg, or betweenabout 3.0×10⁶ cells/kg and about 4.0×10⁶ cells/kg, each inclusive.

58. The method of any of embodiments 1-57, wherein the disease orcondition is a leukemia or lymphoma.

59. The method of any of embodiments 1-58, wherein the disease orcondition is acute lymphoblastic leukemia.

60. The method of embodiment 59, wherein the number of CAR⁺ cells perkilogram administered in the consecutive dose is greater than the numberof CAR⁺ cells per kilogram administered in the first dose.

61. The method of embodiment 60, wherein the number of CAR⁺ cells perkilogram administered in the consecutive dose is at least at or about 2times or at or about 3 times greater than the number of CAR⁺ cells perkilogram administered in the first dose.

62. The method of any of embodiments 1-58, wherein the disease orcondition is a non-Hodgkin lymphoma (NHL).

63. The method of any of embodiments 1-62, wherein the number of CAR⁺cells administered in the first dose is at or about or no more than ator about 1×10⁶ per kilogram of the subject and/or the number of CAR⁺cells administered in the consecutive dose is at or about 3×10⁶ perkilogram of the subject.

64. The method of embodiment 62, wherein the number of CAR⁺ cells perkilogram administered in the consecutive dose is less than or about lessthan or is the same or about the same as the number of CAR⁺ cells perkilogram administered in the first dose.

65. The method of any of embodiments 1-64, further comprisingadministering to the subject one or more additional subsequent doses,wherein the first of said one or more additional subsequent doses isadministered at a time that is at least or greater than 14 days afterthe initiation of the administration of the consecutive dose.

66. The method of embodiment 65, wherein the administration of thefirst, consecutive and subsequent doses comprises administering at leastthree of the doses within at or about 28 days.

67. The method of embodiment 65 or 66, wherein the consecutive dose isadministered at about day 14 following the initiation of administrationof the first dose, one of the at least one additional subsequent dosesis administered at day 28 following the initiation of administration ofthe first dose, and optionally wherein additional subsequent doses areadministered at day 42 and/or day 56 following the initiation ofadministration of the first dose.

68. The method of any of embodiments 1-67, wherein the cells are Tcells.

69. The method of any of embodiments 1-68, wherein the T cells areautologous to the subject.

70. Use of a composition comprising cells expressing a chimeric antigenreceptor (CAR) for manufacture of a medicament for treatment of adisease or condition in a subject previously treated with CAR-expressingcells, wherein:

-   -   the composition is for use 14 to 28 days after the previous        treatment; and/or    -   the composition is formulated for administration of a        consecutive dose in an amount sufficient for reduction in burden        of a disease or condition in the subject having been previously        treated with the CAR-expressing cells.

71. Cells expressing a chimeric antigen receptor (CAR) for use intreating a disease in a subject previously treated with CAR-expressingcells, wherein:

-   -   the cells are for use between about 14 and 28 days after the        previous treatment; and    -   the cells are formulated for administration of a consecutive        dose in an amount sufficient for reduction in burden of a        disease or condition in the subject having been previously        treated with the CAR-expressing cells.

72. The use of embodiment 70 or cells of embodiment 71, wherein thesubject does not exhibit morphologic disease and/or the subject does notexhibit greater than 5% blast cells in the bone marrow.

73. Use of cells expressing a chimeric antigen receptor (CAR) in themanufacture of a medicament for use in a method for treating a diseaseor condition, said method comprising:

-   -   (a) administering to a subject having the disease or condition a        first dose of cells expressing the CAR, said first dose        comprising no more than about 1×10⁶ of the cells per kilogram        body weight of the subject, no more than about 1×10⁸ of the        cells, and/or no more than about 1×10⁸ of the cells/m² of the        subject; and    -   (b) administering to the subject a consecutive dose of cells        expressing a CAR at a time point that is at least or more than        about 14 days after and less than about 28 days after initiation        of said administration in (a).

74. Cells expressing a chimeric antigen receptor (CAR) for use in amethod for treating a disease or condition, said method comprising:

-   -   (a) administering to a subject having the disease or condition a        first dose of cells expressing the CAR, said first dose        comprising no more than about 1×10⁶ of the cells per kilogram        body weight of the subject, no more than about 1×10⁸ of the        cells, and/or no more than about 1×10⁸ of the cells/m² of the        subject; and    -   (b) administering to the subject a consecutive dose of cells        expressing a CAR at a time point that is at least or more than        about 14 days after and less than about 28 days after initiation        of said administration in (a).

75. The use or cells of any of embodiments embodiment 70-74, wherein thecells are formulated for administration in an amount that (i) does notinduce severe CRS in the subject or does not induce CRS in the subject;(ii) does not induce grade 3 or higher neurotoxicity in the subject;(iii) based on clinical data, does not induce severe CRS in a majorityof subjects so-treated; and/or (iv) based on clinical data, does notinduce grade 3 or higher neurotoxicity in a majority of subjectsso-treated.

76. Use of cells expressing a chimeric antigen receptor (CAR) formanufacture of a medicament for the treatment of a disease or conditionin a subject,

-   -   wherein the cells are formulated and/or packaged for        administration to the subject in a first and a consecutive dose        and/or the treatment comprises administering the cells to the        subject in a first and a consecutive dose, wherein:    -   the first dose comprises no more than about 1×10⁶ of the cells        per kilogram body weight of the subject, no more than about        1×10⁸ of the cells, and/or no more than about 1×10⁸ of the        cells/m² of the subject, and    -   the consecutive dose is for administration at a time point (a)        that is at least or more than about 14 days after and less than        about 28 days after initiation of the first administration,        and/or (b) at which (i) the serum level in the subject of a        factor indicative of cytokine release syndrome (CRS) is less        than about 10 times, less than about 25 times, and/or less than        about 50 times that in the subject immediately prior to said        first administration; (ii) the subject does not exhibit grade 3        or higher neurotoxicity; (iii) a CRS-related outcome or symptom        of neurotoxicity in the subject following said administration of        said first dose has reached a peak level and begun to decline        following the first administration, and/or (iv) the subject does        not exhibit a detectable humoral or cell-mediated immune        response against the CAR expressed by the cells of said first        dose.

77. Cells expressing a chimeric antigen receptor (CAR) for use intreatment of a disease or condition in a subject,

-   -   wherein the cells are formulated and/or packaged for        administration to the subject in a first and a consecutive dose        and/or the treatment comprises administering the cells to the        subject in a first and a consecutive dose, wherein:    -   the first dose comprises no more than about 1×10⁶ of the cells        per kilogram body weight of the subject, no more than about        1×10⁸ of the cells, and/or no more than about 1×10⁸ of the        cells/m² of the subject, and    -   the consecutive dose is for administration at a time point (a)        that is at least or more than about 14 days after and less than        about 28 days after initiation of the first administration,        and/or (b) at which (i) the serum level in the subject of a        factor indicative of cytokine release syndrome (CRS) is less        than about 10 times, less than about 25 times, and/or less than        about 50 times that in the subject immediately prior to said        first administration; (ii) the subject does not exhibit grade 3        or higher neurotoxicity; (iii) a CRS-related outcome or symptom        of neurotoxicity in the subject following said administration of        said first dose has reached a peak level and begun to decline        following the first administration, and/or (iv) the subject does        not exhibit a detectable humoral or cell-mediated immune        response against the CAR expressed by the cells of said first        dose.

78. The use or cells of any of embodiments 73-77, wherein the first andconsecutive administrations comprise administering the cells in one ormore unit dose, each unit dose comprising about between 5×10⁷ of thecells and about 5×10⁸ cells, about between 5×10⁷ of the cells and about2.5×10⁸ cells or about between 2.5×10⁸ cells and 4×10⁸ cells; or thecells are formulated in a unit dose comprising no more than about 5×10⁷cell, no more than about 1×10⁸ cells, no more than about 2×10⁸ of thecells, no more than about 2.5×10⁸ of the cells, no more than about3.0×10⁸ of the cells or no more than about 4×10⁸ of the cells.

79. The use or cells of embodiment 78, wherein the first administrationcomprises administering a single unit dose.

80. The use or cells of embodiment 78 or embodiment 79, wherein theconsecutive administration comprises administration of two or more unitdoses.

81. The use or cells of embodiment 78 or embodiment 79, wherein theconsecutive administration comprises administration a single unit dose.

82. The use, composition or cells of any of embodiments 68-81, whereinthe disease or condition is a tumor or a cancer.

83. The use, composition or cells of embodiment 82, wherein the tumor orcancer is leukemia or lymphoma.

84. The use, composition or cells of any of embodiments 68-83, whereinthe consecutive dose is formulated for administration of an increasednumber of CAR-expressing cells as compared to the first dose or previousdose.

85. The use, composition or cells of embodiment 84, wherein thecomposition is for use in treating acute lymphoblastic leukemia.

86. The use, composition or cells of any of embodiments 68-83, whereinthe consecutive dose is formulated for administration of less than orabout the same number of CAR-expressing cells as the number ofCAR-expressing cells in the previous dose.

87. The use, composition or cells of embodiment 84 or embodiment 86,wherein the composition is for use in treating non-Hodgkin lymphoma(NHL).

88. An article of manufacture, comprising:

a plurality of sealable containers, each individually comprising a unitdose of cells expressing a chimeric antigen receptor (CAR) foradministration to a subject, said unit dose comprising about 1×10⁸ ofthe cells, no more than about 1×10⁸ of the cells, about 5×10⁷ of thecells, no more than about 5×10⁷ of the cells, about 1×10⁶ cells per kgof the subject, or no more than about 1×10⁶ of the cells per kg of thesubject;

-   -   packaging material; and    -   a label or package insert comprising instructions for        administering a plurality of said unit doses to the subject by        carrying out a first administration and a consecutive        administration, said first administration comprising delivering        one of said unit doses to the subject and said consecutive        administration comprising administering one or a plurality of        said unit doses to the subject.

89. The article of manufacture of embodiment 88, wherein theinstructions specify that said consecutive administration is to becarried out at a time between about 14 or 15 and 27 days, optionally atabout day 17 or about day 21, following said first administration.

90. The article of manufacture of embodiment 88 or embodiment 89,wherein the instructions specify that said consecutive administration isto be carried out at a time after which it has been determined that aserum level of a factor indicative of cytokine-release syndrome (CRS) inthe subject is less than about 10 times, less than about 25 times,and/or less than about 50 times the serum level of said indicator in thesubject immediately prior to said first administration.

91. The article of manufacture of any of embodiments 88-90, wherein theinstructions specify that said consecutive administration is to becarried out at a time after which it has been determined that anindicator of CRS has peaked and is declining, and/or that the subjectdoes not exhibit a detectable adaptive host immune response specific forthe CAR expressed by the cells of said first dose.

92. The article of manufacture of any of embodiments 88-91, wherein thecells have been derived from the subject.

93. The article of manufacture of any of embodiments 88-92, wherein saidlabel and/or said packaging material further comprises an identifierspecific to the subject, indicating that the cells were derived from thesubject and/or should be administered to the subject specifically.

94. The article of manufacture of any of embodiments 88-93, wherein thecontainers are or comprise flexible cell infusion bags.

95. A method of treatment, comprising:

-   -   a) assessing a factor indicative of disease burden in a subject        having or suspected of having a tumor; and    -   b) based on the result of the assessment, determining the dose        of cells expressing a chimeric antigen receptor (CAR) to be        administered to the subject, wherein:        -   i) if the assessment determines that the subject does not            have morphologic disease or does not have substantial            morphologic disease or does not have a disease burden at or            above a threshold level, administering to the subject a dose            of CAR-expressing cells greater than 1×10⁶ cells/kg; and        -   ii) if the assessment determines that the subject has            morphologic disease or substantial morphologic disease or a            disease burden at or above a threshold level, administering            to the subject a dose of CAR-expressing cells that is            relatively lower as compared to in ii).

96. The method of embodiment 95, wherein:

-   -   the subject does not exhibit morphologic disease or substantial        morphologic disease or a disease burden at or above a threshold        level and is administered a dose of cells that is greater than        or about 2×10⁶ cells/kg, 3×10⁶ cells/kg, 4×10⁶ cells/kg, 5×10⁶        cells/kg, 6×10⁶ cells/kg, 7×10⁶ cells/kg, 8×10⁶ cells/kg, 9×10⁶        cells/kg, 1×10⁷ cells/kg or 2×10⁷ cells/kg; and/or    -   the subject exhibits morphologic disease or substantial        morphologic disease or has a disease burden above a threshold        level and is administered a dose of cells that is less than or        less than about 1×10⁶ cells/kg or 5×10⁶ cells/kg.

97. The method of embodiment 95 or embodiment 96, wherein the subjectexhibits morphologic disease and/or substantial morphologic diseaseand/or the disease burden is at or above a threshold level if there aregreater than or equal to or about 5% blasts in the bone marrow, greaterthan or equal to or about 10% blasts in the bone marrow, greater than orequal to or about 15% blasts in the bone marrow or greater than or equalto or about 20% blasts in the bone marrow.

98. The method of any of embodiments 95 to 97, wherein:

-   -   if the subject exhibits less than or less than about 5% blasts        the subject is administered a dose of cells of greater than or        about 1×10⁶ cells/kg 2×10⁶ cells/kg, 3×10⁶ cells/kg, 4×106        cells/kg, 5×10⁶ cells/kg, 6×10⁶ cells/kg, 7×10⁶ cells/kg, 8×10⁶        cells/kg, 9×10⁶ cells/kg, 1×10⁷ cells/kg or 2×10⁷ cells/kg        and/or if the subject exhibits greater than or equal to or about        5% blasts the subject is administered a dose of cells that is        less than about 1×10⁶ cells/kg or 5×10⁶ cells/kg;    -   if the subject exhibits less than or less than about 10% blasts        the subject is administered a dose of cells of greater than or        about 1×10⁶ cells/kg 2×10⁶ cells/kg, 3×10⁶ cells/kg, 4×10⁶        cells/kg, 5×10⁶ cells/kg, 6×10⁶ cells/kg, 7×10⁶ cells/kg, 8×10⁶        cells/kg, 9×10⁶ cells/kg, 1×10⁷ cells/kg or 2×10⁷ cells/kg        and/or if the subject exhibits greater than or equal to or about        10% blasts the subject is administered a dose of cells that is        less than about 1×10⁶ cells/kg or 5×10⁶ cells/kg;    -   if the subject exhibits less than or less than about 15% blasts        the subject is administered a dose of cells of greater than or        about 1×10⁶ cells/kg 2×10⁶ cells/kg, 3×10⁶ cells/kg, 4×10⁶        cells/kg, 5×10⁶ cells/kg, 6×10⁶ cells/kg, 7×10⁶ cells/kg, 8×10⁶        cells/kg, 9×10⁶ cells/kg, 1×10⁷ cells/kg or 2×10⁷ cells/kg        and/or if the subject exhibits greater than or equal to or about        15% blasts the subject is administered a dose of cells that is        less than about 1×10⁶ cells/kg or 5×10⁶ cells/kg; if the subject        exhibits less than or less than about 20% blasts the subject is        administered a dose of cells of greater than or about 1×10⁶        cells/kg 2×10⁶ cells/kg, 3×10⁶ cells/kg, 4×10⁶ cells/kg, 5×10⁶        cells/kg, 6×10⁶ cells/kg, 7×10⁶ cells/kg, 8×10⁶ cells/kg, 9×10⁶        cells/kg, 1×10⁷ cells/kg or 2×10⁷ cells/kg and/or if the subject        exhibits greater than or equal to or about 20% blasts the        subject is administered a dose of cells that is less than about        1×10⁶ cells/kg or 5×10⁶ cells/kg.

99. The method of any of embodiments 95 to 98, wherein the tumor is aleukemia or lymphoma, optionally a B cell-derived leukemia or lymphoma.

100. The method of any of embodiments 95 to 99, wherein the subject doesnot exhibit cytokine release syndrome (CRS), does not exhibit severeCRS, does not exhibit neurotoxicity, does not exhibit severeneurotoxicity, or does not exhibit neurotoxicity above grade 3 followingadministration of the dose of cells.

101. The method of any of embodiments 95 to 100, wherein theadministration in (a) leads to a reduction in burden of the disease orcondition in the subject, as indicated by a reduction in one or morefactors indicative of disease burden following said administration in(a).

102. The method of any of embodiments 95 to 101, further comprisingadministering one or more consecutive doses to the subject.

EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Treatment of Cancer Patients with First and Consecutive Dosesof CAR-Expressing Autologous T Cells

T cells are isolated from peripheral blood of human subjects with cancerby immunoaffinity-based enrichment and the cells cultured and transducedwith viral vectors encoding a chimeric antigen receptor (CAR) thatspecifically binds to an antigen expressed by the cancer in the subject,which is a tumor-associated or tumor-specific antigen. The cells arecryopreserved in infusion medium in individual flexible infusion bags,each containing a single unit dose of the cells, which is about 1×10⁶cells per kilogram body weight of the subject or about 5×10⁵ cells perkilogram body weight of the subject. The cells are maintained at atemperature below −130° C. prior to infusion.

Prior to initiation of cell therapy, blood is obtained from the subjectsand, optionally, the levels of one or more serum factors indicative ofcytokine release syndrome (CRS) such as tumor necrosis factor alpha(TNFα), interferon gamma (IFNγ), and IL-6, are assessed in the serum byELISA. Tumor burden is optionally assessed by measurement of the size ormass of a solid tumor, such as by PET or CT scan, or by assessing thenumber of cells of the patient associated with the cancer, such as inthe bone marrow or peripheral blood, before treatment begins.

The cells are thawed at bedside by warming to approximately 37° C. andsubjects administered a first dose of the cells by single infusion. Theamount of the first dose is a single unit dose. For subjects deemed tohave low tumor burden, two unit doses may be administered in the firstdose. The first dose is administered intravenously (IV) by continuousinfusion, over a period of approximately 15 to 30 minutes.

Following administration of the first dose, the subjects receivephysical examinations and are monitored for any symptoms of toxicity ortoxic outcomes, such as fever, hypotension, hypoxia, neurologicdisturbances, or an increased serum level of an inflammatory cytokine orC reactive protein (CRP). Optionally, following administration of thefirst dose, on one or more occasions, blood is obtained from thepatients and the levels of serum factors indicative of CRS are assessedby ELISA. The levels of the serum factors are compared to those obtainedimmediately prior to administration of the first dose. If necessary,anti-IL6 or other CRS therapy is administered to reduce signs of CRS.

The presence or absence of an anti-CAR immune response in the subject isoptionally detected following the administration of the first dose, forexample, at 1, 2, 3, and/or 4 weeks following the initiation of theadministration, for example, by ELISA, ELISPOT, cell-based antibodyassay, and/or mixed-lymphocyte reaction.

The percent reduction in tumor burden achieved by the first dose isoptionally measured on one or more occasions following administration ofthe first dose by scans, such as PET and CT scans, in patients withsolid tumors, and/or by quantifying disease-positive cells in blood ortumor sites, e.g., in subjects with hematological cancers, and comparingthe values to those observed immediately prior to the first dose.

A consecutive dose is administered. In some subjects, the consecutivedose is administered in 21 days following the initiation of theadministration of the first dose. In some cases, the consecutive dose isonly administered if the level of a tested CRS-related outcome or serumfactor is below an acceptable level and if no anti-CAR immune responseis detected in the subject at 21 days following the first doseadministration. In other subjects, the consecutive does is administeredat a time that is greater than 3 days following the administration ofthe first dose, and at which the subject is deemed to not have CRS orsevere CRS, or at which levels of all tested serum factors indicative ofCRS are below 20% of that observed at a peak following the first doseadministration, and the subject is deemed not to have a detectableanti-CAR immune response.

The size of the consecutive dose is patient-specific, and is based ontumor burden, presence of an anti-CAR immune response, and level ofCRS-related outcomes. Some patients are administered a consecutive dosecontaining 1, 2, 3, or even more unit doses of the cells. Theconsecutive dose is administered by continuous infusion, IV, overapproximately 15 to 30 minutes.

Beginning after the first dose and continuing for up to several years,the subjects are monitored on a regular basis. Development of ananti-CAR immune response is assessed and tumor burden is measured.Optionally, during follow-up visits, the CAR-expressing cells aredetected by flow cytometry and quantitative polymerase chain reaction(qPCR) to measure in vivo proliferation and persistence of theadministered cells.

Example 2 Assessment of Neurotoxicity of CAR-T Cell Treatment inSubjects Having Morphological Disease Prior to Treatment

Subjects with CD19⁺ B cell acute lymphoblastic leukemia (ALL) wereadministered autologous T cells expressing an anti-CD19 chimeric antigenreceptor (CAR). The CAR included a truncated EGFR (EGFRt) portion as amarker. Prior to administration of the cells, patients underwentleukapheresis and were treated with chemotherapy. To generate theautologous CAR-expressing T cells, T cells were isolated byimmunoaffinity-based enrichment from leukapheresis samples fromindividual subjects, activated and transduced with a viral vectorencoding an anti-CD19 CAR. The cells were expanded, frozen, and thawedat bedside prior to administration.

Tumor burden was assessed prior to treatment by evaluating bone marrowand the percent of bone marrow blasts was determined. Subjects having atleast 5% blasts in bone marrow were deemed to have morphological disease(MD). Subjects exhibiting complete remission (CR) as defined below,including having less than 5% blasts in the bone marrow, but showingmolecularly detectable disease in the bone marrow (by flow cytometry)were deemed to have minimal residual disease (MRD). CAR-expressing Tcells were administered to subjects by single intravenous (IV)continuous infusion, over approximately 15-30 minutes, at varying dosesranging from about or approximately 0.9×10⁶ CAR+ cells/kg to about orapproximately 5.6×10⁶ CAR+ cells/kg (see FIG. 1A-C). Cyclophosphamidewas administered to subjects as a preconditioning chemotherapeutictreatment 2 to 7 days before cell infusion.

Disease status of subjects (MRD or MD) was assessed subsequent toadministration of CAR-expressing T cells to assess response totreatment. Complete remission (CR) was determined in subjects if therewas a restoration of normal hematopoiesis with neutrophil count>1,000×10⁶/L, platelet count of >100,000×10⁶/L and hemoglobin >10 g/dL;<5% blasts present in a post-treatment bone marrow differential; and noclinical evidence of leukemia for a minimum of four weeks. Followingtreatment, subjects also were assessed and monitored for neurotoxicity(neurological complications including symptoms of confusion, aphasia,seizures, convulsions, lethargy, and/or altered mental status), gradedbased on severity (using a Grade 1-5 scale (see, e.g., Guido Cavaletti &Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010),with grade 3 (severe symptoms), 4 (life-threatening symptoms) or 5(death) being deemed severe neurotoxicity. Cytokine release syndrome(CRS) also was determined and monitored, graded based on severity.

Response, presence of severe CRS, and presence of severe neurotoxicityfollowing treatment with a single infusion of varying doses ofCAR-expressing T cells, compared to dosage, were assessed in groups ofsubjects separated based on disease burden prior to treatment. Resultsare shown in FIGS. 1A-C.

As shown in FIG. 1A, complete remission (CR) was observed in themajority of subjects treated with CAR-expressing T cells at all dosestested, regardless of disease burden (morphological or molecularlydetectable disease) prior to treatment. CRS was observed primarily insubjects classified as having morphological disease prior to treatment.The results depicted in FIG. 1B, however, show that within this group,the presence of CRS was observed for subjects having received variousdosage levels of CAR-expressing T cells.

As shown in FIG. 1C, severe neurotoxicity also was most frequentlyobserved in subjects with morphological disease as composed to minimalresidual disease prior to treatment in contrast to the results for CRS,however, neurotoxicity was observed only in those subjects receivinghigher doses. Subjects analyzed in this study who had received a dose ofCAR-expressing T cells lower than about 3.5×10⁶ cells/kg, did notexhibit severe neurotoxicity after treatment. With one exception, severeneurotoxicity was generally not observed in subjects classified ashaving molecularly detectable disease prior to treatment. Thus, subjectsreceiving a lower dose of administered T cells and/or who had a lowerdisease burden prior to treatment did not generally exhibit severeneurotoxicity.

These results support, in order to minimize toxicity and maximizeefficacy, using a dosage regime that includes administering to subjects,including those with morphologic disease, a first low dose ofCAR-expressing T cells (which may not necessarily be sufficient toeradicate disease in all or most subjects) to reduce disease burden,followed by a consecutive or subsequent dose of cells after tumor burdenhas been reduced. The results also support a conclusion that if desiredor needed in a particular disease or context (for example, to promote anincreased response or efficacy), the consecutive or subsequentadministration of cells (given after reduction in tumor burden, at whichpoint all or most subjects should exhibit minimal disease), theconsecutive dose can be carried out at a higher dose, without or withminimal risk of severe neurotoxicity.

Example 3 Assessment of Efficacy and Toxicity Following First andConsecutive Doses of CAR⁺ Autologous T Cells

Treatment efficacy and toxic effects were assessed in a subset ofsubjects in the study described in Example 2 who were administeredmultiple doses of the CAR⁺ T cells. Table 3 below sets forth theparticular dosages of cells administered in the various multiple doses,and in each case, the time that elapsed between the first andconsecutive dose. Table 3 also sets forth each subject's tumor burden(MRD or MD with % of blast cells in bone marrow; listed under the columnlabeled “Disease burden”) as assessed prior to administration of eachdose, based on criteria as described in Example 2. Table 3 also listsresults for each patient for response to each administration (under thecolumn “Response,” listing the tumor burden following administration,which as compared to the pre-administration disease burden, indicatesresponse to treatment), presence or absence (Y/N) of severe CRS andsevere neurotoxicity as described in Example 2.

TABLE 3 Safety and Efficacy of First and Consecutive CAR⁺ T Cell DosesFirst Dose Consecutive Dose Severe Days Severe Disease Dose × SevereNeuro- between Disease Dose × Severe Neuro- ID burden 10⁶/kg CRStoxicity Response doses burden 10⁶/kg CRS toxicity Response 1 MRD+ 1.74N N MRD− 133 MRD+ 1.37 N N MRD− [2%] CR [2%] CR 2 6% 3.08 N N MRD− 86MRD+ 1.50 N N MRD− CR [3%] CR 2* 90% 4.27 N N 48% 23 56% 3.27 N N NR 4MRD+ 0.47 N N MRD+ 203 MRD+ 3.84 N N MRD+ [4%] CR [3%] 5 97% 2.99 N N NR15 75% 3.80 N N NR 6 60% 3.48 N Y MRD− 414 MRD+ N/A N N MRD− CR [2%] CR7 MRD+ 4.15 N Y MRD+ 103 MRD+ 4.53 N N MRD− CRi 8 85% 4.10 Y Y MRD− 10415% 4.27 N Y MRD− CR CR 9 25% 1.79 N N MRD+ 79 87% 1.98 N N CR [2%]*3^(rd) dose; subject in CR following 1^(st) and 2^(nd) doses ID:Subject Number MRD: Minimal Residual Disease CR: complete remission Cri:complete remission with incomplete blood count recovery NR: No responseN/A: not available N: No Y: Yes

The results in Table 3 show that a majority of treated subjects wereresponsive to the first dose of cells as evidenced by a reduction intumor burden in the subject, for example, reduction in tumor burden frompresence of >5% of blast cells to MRD⁺ or in some cases MRD⁻, or fromMRD to MRD⁻. Complete remission (CR) of some subjects also was observed.As shown in Table 3, tumor reduction also was observed for subjectsfollowing administration of a consecutive dose of cells as evidenced bycomplete remissions or disease reduction as assessed from flowcytometric analysis for presence of MRD (see patient ID No. 1, 2, 7, 8and 9). Thus, this result demonstrates that administration of theconsecutive dose of cells also was efficacious. Most subjects did notexhibit severe CRS at any of the doses. Further, consistent with Example2 severe neurotoxicity was observed to occur following administrationonly in subjects who had received higher doses of CAR-expressing cellsand generally only in subjects with morphologic disease.

In some cases, Table 3 demonstrates that some subjects were notresponsive (NR) to the first dose (see patient ID No. 5 and ID No. 2*)so that a reduction in disease or tumor burden levels was not achieved.Further, the results show that such failure to achieve clinicalremission after infusion of the first dose did not increase the risk orfrequency of neurotoxicity in the subject when administered aconsecutive higher dose, since severe neurotoxicity was not detectedafter receiving a consecutive higher dose in either of these subjects.

In some embodiments, lack of (or relatively lower) degrees ofresponsiveness or toxicity in a subject indicates that the subject maynot have responded well to CAR⁺ T cell therapy, but is also not at riskfor certain toxic adverse events upon another infusion, which mayotherwise indicate a need to avoid higher doses for subsequentadministration. Therefore, these results support a dosage regime (e.g.,if desired or necessary for maximizing or improving efficacy) in which afirst low dose administration is followed (e.g., in a relatively shorttime) by a consecutive administration, carried out using a higher doseas compared to the first dose. The results presented here show that evenif certain subjects do not respond well to the first dose (and thus donot have low tumor burden at the time of the consecutive dose) the riskof severe neurotoxicity is low upon administration of the consecutivedose, even for these non-responding subjects.

To further assess efficacy of cells administered in the first andconsecutive dose, biological activity of administered cells was comparedbased on peak levels of measurements that were taken of serum levels ofC reactive protein (CRP). Elevated peak levels of CRP in serum weretaken as evidence of T cell expansion following infusion. The resultsare set forth in FIG. 2. The results show evidence of cell expansionafter infusion of both a first and consecutive dose of T cells. Thus,these results show that, while subjects exhibited reduced CRS andneurotoxicity following a consecutive as opposed to a firstadministration of CAR+ T cells, the biological activity of the cellsadministered in and their ability to expand was comparable in eachadministration.

Subjects in the study also received physical examinations and weremonitored for symptoms of other adverse events (AE), including those setforth in Table 4. To assess differences between first and consecutiveadministrations of CAR+ T cells in subjects, these data were compared. Atreatment emergent AE (TEAE) after infusion of the first dose wasdefined as any AE occurring within 30 days after receiving the firstdose but prior to receiving the consecutive dose. A TEAE after infusionof the consecutive dose was defined as any AE occurring within 30 daysafter receiving the consecutive dose but prior to any subsequent dose.Table 4 sets forth the number of subjects that exhibited a TEAE and thepercentage (in parenthesis) of subjects exhibiting such TEAE compared tothe number of total subjects assessed for the same TEAE. The resultsshow that the percentage of adverse events was comparable followinginfusion of the first dose and consecutive dose.

TABLE 4 Adverse Events TEAE post- TEAE post- infusion dose infusion doseAdverse Event #1 (n = 33) #2 (n = 11) All 30 (90.9) 8 (72.7) Febrileneutropenia 15 (45.5) 2 (18.2) Abdominal pain 3 (9.1) 1 (9.1) Nausea 8(24.2) 1 (9.1) Chills 10 (30.3) 1 (9.1) Pyrexia 12 (36.4) 3 (27.3)Cytokine release syndrome 5 (15.2) 1 (9.1) (CRS) Alanineaminotransferase 3 (9.1) 1 (9.1) increased Aspartate aminotransferase 2(6.1) 1 (9.1) increased Hyperglycaemia 5 (15.2) 1 (9.1)Hypophosphataemia 14 (42.5) 1 (9.1) Hypophosphataemia 14 (42.5) 1 (9.1)Muscular weakness 3 (9.1) 1 (9.1) Convulsion 6 (18.2) 1 (9.1) Dizziness2 (6.1) 1 (9.1) Encephalopathy 9 (27.3) 1 (9.1) Dermatitis acneiform 0(0.0) 1 (9.1) Hypotension 12 (36.4) 1 (9.1)

Example 4 Impact of CAR⁺ T Cell Dose on Overall Survival of DifferentSubject Populations

To further assess the impact of CAR⁺ T cell dose on overall treatment ofsubjects, overall survival of subjects in the study described in Example2 was compared between small groups of subjects, separated based uponnumber of cells administered and disease state at the time ofadministration. Product-limit survival was determined by calculating theKaplan-Meier estimate with censored observations, such as occurred, forexample, if a patient withdrew from the study.

Comparing all subjects in a group of subjects who had been administereda dose of fewer than 2.5×10⁶ CAR⁺ cells/kg and all subjects in anothergroup of subjects who had been administered a dose with greater than2.5×10⁶ CAR⁺ T cells/kg, the results indicated an overall survivaladvantage for the group of subjects administered the higher dose.Looking just at subjects having morphologic disease at the time ofadministration, the observed effect on overall survival with a higherdose was even greater. Collectively, the results presented hereinsupport a dosage regime in which a higher dose (as compared to a firstdose) is used in a consecutive administration, when the consecutive doseis given at a time at which disease burden remains reduced in patientson average, but at which the risk of CRS and/or neurotoxicity remainslow. The results in this example support a conclusion that use of ahigher consecutive dose will promote increased efficacy. Moreover, asnoted, the results presented in Example 2 support a conclusion that useof a higher dose upon subsequent administration (at which subjectsshould have low disease burden or be otherwise not at risk for toxicity)will not lead to an increased toxicity risk.

Example 5 Multiple Dose Regimen of CAR⁺ T Cells for Treating AcuteLymphoblastic Leukemia (ALL)

In an exemplary dose regimen, subjects with CD19⁺ B cell acutelymphoblastic leukemia (ALL) were treated with two doses ofCAR-expressing T cells, which included administering a first low dose ofcells and a consecutive higher dose of cells. Before treatment,autologous CAR-expressing T cells were generated substantially asdescribed in Example 2. Subjects received preconditioning chemotherapyincluding a single intravenous lymphodepleting dose of 1.0-3.0 g/m²cyclophosphamide at 2-5 days prior to the first dose of CAR-expressing Tcells. The first dose included approximately 1×10⁶ cells/kg patientweight. A consecutive dose of cells expressing the CAR (approximately3×10⁶ such cells/kg patient weight) was administered 14-28 days afterthe first dose at a dose.

Subjects were monitored for efficacy of treatment, including by bonemarrow, peripheral blood, and cerebrospinal fluid (CSF) examination,evaluation of central nervous system (CNS) symptoms, in order to assessand monitor disease burden (including levels and presence or absence ofmorphologic and degree of molecularly-detectable disease), evidence ofadverse events, including CRS and neurotoxicity, and survival.

Example 6 Repeated Dosing Schedule of CAR⁺ T Cells for Treating AcuteLymphoblastic Leukemia (ALL)

In an exemplary dose regimen, subjects with relapsed or refractory Bcell acute lymphoblastic leukemia (ALL) are treated with repeated dosesof CAR-expressing T cells, which includes administering at least threedoses of cells within the first 28 days. Before treatment, autologousCAR-expressing T cells are generated substantially as described inExample 2. Optionally, subjects receive preconditioningimmunosuppressive chemotherapy of cyclophosphamide and/or fludarabine(CY/FLU), which is administered at least two days before the first doseof CAR-expressing cells and generally no more than 5 or no more than 7days before administration of cells.

Subjects receive a first dose of CAR-expressing cells that is less thanor equal to about 1×10⁶ cells/kg patient weight, such as ranging fromabout 0.4×10⁶ cells/kg to about 1×10⁶ cells/kg, inclusive. Within 14-28days after administration of the first dose, and/or prior to developmentof an immune response to the CAR, subjects are infused with twoadditional higher doses of cells. In some embodiments, a consecutivedose of CAR-expressing cells is administered about 14 days after thefirst dose at a dose that is higher than the first dose, such as a doseranging from about 2.5×10⁶ cells/kg to about 4.5×10⁶ cells/kg, such asapproximately 3×10⁶ cells/kg patient weight, followed by a third dose ofCAR-expressing cells that is administered about 28 days after the firstdose at a dose that is higher than the first dose, such as a doseranging from about 2.5×10⁶ cells/kg to about 4.5×10⁶ cells/kg, such asapproximately 3×10⁶ cells/kg patient weight. In some embodiments, one ormore subsequent doses of cells are administered. In some embodiments, afourth dose of CAR-expressing cells is administered within about 42 to56 days after the first dose at a dose that is higher than the firstdose, such as a dose ranging from about 2.5×10⁶ cells/kg to about4.5×10⁶ cells/kg, such as approximately 3×10⁶ cells/kg patient weight.

Subjects are monitored for efficacy of treatment by measuring theoverall remission rate (ORR) after the final dose of cells in subjects.In some embodiments, efficacy is monitored in subjects with morphologicevidence of disease prior to treatment (greater than or equal to 5% ofcells in bone marrow were blasts). ORR is determined as the proportionof subjects with CR with incomplete blood count recovery (CRi), asdetermined by examination of the bone marrow, peripheral blood, andcerebrospinal fluid (CSF), as well as physical examination andevaluation of central nervous system (CNS) symptoms.

Example 7 Multiple Dose Regimen of CAR⁺ T Cells with LymphodepletingChemotherapy Pre-Conditioning for Treating Non-Hodgkin Lymphoma (NHL)

In exemplary dose regimens for treating CD19⁺ B cell Non-HodgkinLymphoma (NHL), a multiple dose regimen of CAR-expressing T cells isused to treat subjects. Before treatment, autologous CAR-expressing Tcells are generated substantially as described in Example 2. Subjectswith NHL are treated with at least two doses of CAR-expressing T cells.Subjects receive a first dose of CAR-expressing cells that is less thanor equal to about 1×10⁶ cells/kg patient weight, such as ranging fromabout 0.4×10⁶ cells/kg to about 1×10⁶ cells/kg, inclusive. In exemplarydosage regimes, a consecutive dose of CAR-expressing T cells isadministered 14 to 28 days after the first dose.

In one exemplary dosage regime, subjects with NHL receive a consecutivedose of CAR-expressing at a dose that is the same or lower than thefirst dose of CAR-expressing T cells, such as a dose that is less thanor equal to about 1×10⁶ cells/kg patient weight, such as ranging fromabout 0.4×10⁶ cells/kg to about 1×10⁶ cells/kg, inclusive. Optionally,further repeat doses are administered 14 to 28 days after a prioradministration at a dose that is less than or equal to the doseadministered in the prior administration.

In one exemplary dosage regime, prior to receiving a consecutive dose,subjects are optionally monitored for tumor burden. If molecularremission is detected, as evidenced by tumor reduction frommorphological disease to MRD, subjects are administered a consecutivedose of CAR-expressing T cells at a dose that is higher than the firstdose, such as a dose ranging from about 2.5×10⁶ cells/kg to about4.5×10⁶ cells/kg, such as approximately 3×10⁶ cells/kg patient weight.If molecular remission has not occurred, subjects are administered aconsecutive dose of CAR-expressing T cells at a dose that is the same orlower than the first dose of CAR-expressing T cells, such as a dose thatis less than or equal to about 1×10⁶ cells/kg patient weight, such asranging from about 0.4×10⁶ cells/kg to about 1×10⁶ cells/kg, inclusive.Optionally, further repeat doses are administered 14 to 28 days after aprior administration, with higher doses being administered if molecularremission has occurred and lower doses being administered if molecularremission has not occurred.

In one exemplary dosage regime, prior to receiving the first dose,subjects receive an immunodepleting preconditioning chemotherapy ofcyclophosphamide and fludarabine (CY/FLU), which is administered atleast two days before the first dose of CAR-expressing cells andgenerally no more than 7 days before administration of cells. Afterpreconditioning treatment, subjects are administered the first dose asdescribed above at a dose of CAR-expressing T cells that is less than orequal to about 1×10⁶ cells/kg patient weight, such as ranging from about0.4×10⁶ cells/kg to about 1×10⁶ cells/kg, inclusive. Subjects areadministered a consecutive dose of CAR-expressing T cells at a dose thatis higher than the first dose, such as a dose ranging from about 2.5×10⁶cells/kg to about 4.5×10⁶ cells/kg, such as approximately 3×10⁶ cells/kgpatient weight. Optionally, further repeat doses are administered 14 to28 days after a prior administration at a dose ranging from about2.5×10⁶ cells/kg to about 4.5×10⁶ cells/kg, such as approximately 3×10⁶cells/kg patient weight.

Example 8 Assessment of PD1/PD-L1 Expression In T-Cells StimulatedThrough a Chimeric Antigen Receptor (CAR)

T cells were isolated by immunoaffinity-based enrichment fromleukapheresis samples from human subjects, and cells were activated andtransduced with a viral vector encoding an anti-CD19 chimeric antigenreceptor (CAR) containing a human CD28-derived intracellular signalingdomain and a human CD3 zeta-derived signaling domain. Surface expressionon the resulting isolated compositions (of the CAR and of certain T cellmarkers) was assessed by flow cytometry, to determine, in thecomposition, the percentage of CAR+ cells among all T cells in the andamong T cell subsets, as well as ratio of CD4+ to CD8+ T cells (seeTable 5).

TABLE 5 Anti-CD19 CAR Expression on Transduced T cells CD3+CAR+ CD4+CAR+CD8+CAR+ CD3+CD4+ CD3+CD8+ percent 49.91 23.60 28.73 40.03 53.66(average) Standard 2.97 1.18 2.38 1.10 1.22 Deviation

The composition then was subdivided into different samples by incubationwith: 1) K562 cells expressing the antigen for which the CAR wasspecific (K562-tCD19 cells) (antigen-specific coculture); 2) K562 cellsexpressing an unrelated antigen (K562-ROR1 cells) (non-specificcoculture control); or 3) plate-bound anti-CD3 antibody and solubleanti-CD28 antibody (for stimulation via the TCR complex), initiallyusing plate-bound anti-CD3 and soluble anti-CD28, and at day 3, whereapplicable, incubation. For (1) and (2), K562 (immortalized myelogenousleukemia line) cells, were engineered to express CD19 and ROR1,respectively, and incubated with the CAR-expressing T cells at a 1:1ratio. For each of the conditions, CAR-expressing T cells werestimulated for 24 hours. An unstimulated sample (“media,” no K562 cellsor stimulating antibodies) was used as an additional negative control.

After 24 hours in culture, flow cytometry was performed to assesssurface expression of PD1, PD-L1, PD-L2, T cell markers, and CAR (basedon goat-anti-mouse (“GAM”) staining to detect the murine variable regionportion of the CAR) on the cells in each sample. Live, single cells withforward scatter and side scatter profiles matching lymphocytes weregated for analysis. Expression of PD1, PD-L1 and PD-L2 was assessed onvarious gated populations of T cells (CD4⁺/CAR⁺, CD4⁺/CAR⁻, CD8⁺/CAR⁺,and CD8⁺/CAR⁻), with gates set based on the surface expression ofvarious markers, and using values for the negative control (“media”)sample to determine appropriate gating.

As shown in FIGS. 3A and 4A, PD1 and PD-L1 expression increased withintwenty-four (24) hours in both CD4⁺/CAR⁺ and CD8⁺/CAR⁺ T cells whencultured with cells expressing the antigen to which the CAR was specific(K562-tCD19). This increase in expression of PD1 and PD-L1 was notobserved within this timeframe in CAR+ cells incubated with cells of thesame type expressing an irrelevant antigen cells (K562-ROR1) or in anyof the CD4⁺ or CD8⁺ cell populations incubated under conditions designedto effect stimulation through the TCR complex (anti-CD3 and anti-CD28antibodies). Expression of PD-L2 was not upregulated within thistimeframe under any of the stimulated conditions tested.

As shown in FIGS. 3B and 4B, the increase in expression of PD-1 andPD-L1 in cells incubated with CD19-expressing cells was observed to beprimarily due to expression of the anti-CD19 CAR. Neither the CD4+-gatednor the CD8+-gated T cells that did not express the CAR (“CAR-”)exhibited substantial increases in PD-1 or PD-L1 surface expressionfollowing incubation with the CD19-expressing cells.

Similar results were obtained in the presence of T cells geneticallyengineered with an anti-CD19 chimeric antigen receptor (CAR) containinga human 4-1BB-derived intracellular signaling domain and a human CD3zeta-derived signaling domain. Thus, the results showed that theupregulation of PD-1 and PD-L1 occurred on T cells transduced with CARconstructs containing either a CD28 or 4-1BB costimulatory signalingdomain. These data demonstrate upregulation of surface expression of PD1and PD-L1 within twenty-four hours following stimulation through thechimeric antigen receptor, but not following stimulation underconditions designed to mimic signal through the canonical T cell antigenreceptor complex (CD3/CD28 antibodies).

These data further support a conclusion that exposure, in vivo, uponadministration to subjects, to antigen to which the CAR was specific mayin some contexts result in upregulation of PD-L1 and/or PD-1, which mayoccur to a greater degree and/or more rapidly than (or in distinctionfrom) what would occur following interaction with cognate antigenthrough an endogenous TCR.

Upregulation of such molecules and other inhibitory markers maycontribute to loss of function and/or exhaustion of the T cells and forexample may impair long-term exposure to the cells. A repeat orconsecutive dose(s) of cells may be used to deliver cells not yetexpressing the inhibitory molecules, such as PD-1 and/or PD-L1, orexpressing them at lower levels compared to the cells present in thesubject. Thus, these data provide further support for a multiple dosingschedule, in which a consecutive dose of T cells is administered to asubject following an initial dose, such as at a time at which PD-L1 orPD-1 has been or is upregulated on cells of the initial dose, followingexposure to the target antigen.

In some embodiments, in the consecutive dose, the inhibitory molecule(s)are not expressed or substantially expressed (or expressed to the samedegree as a reference cell population) on the cells therein (or ongreater than 50, 40, 30, 20, 10, or 5% of the cells therein). In someembodiments, repeated doses of cells that do not express or do notsubstantially express inhibitory molecules, such as PD-1 and PD-L1, canextend the time during which functional CAR-expressing T cells orCAR-expressing T cells having robust function are present in thesubject. In some embodiments, replenishing the army of geneticallyengineered T cells by administering one or more consecutive doses canlead to a greater and/or longer degree of exposure to the antigenreceptor (e.g. CAR)-expressing cells and improve treatment outcomes. Insome embodiments, the consecutive dose is administered at a time atwhich PD-L1 or PD-1 is upregulated compared to a reference level orpopulation, such as compared to the cells in the composition of thefirst dose immediately prior to administration to the subject, forexample, to a degree that is at least 10, 20, 30, 40, 50, 60, 70, or 80%higher surface expression as compared to the reference population.

Example 9 Assessment of Neurotoxicity in Subjects Based on Tumor Burden

Subjects with CD19⁺ B cell acute lymphoblastic leukemia (ALL) wereadministered autologous T cells expressing an anti-CD19 chimeric antigenreceptor (CAR). Before treatment, autologous CAR-expressing T cells weregenerated substantially as described in Example 2

Neurotoxicity was observed in a cohort of patients after receiving asingle IV continuous infusion of CAR-expressing T cells, at a dose ofeither 2×10⁵ cells/kg (N=13), 2×10⁶ cells/kg (N=15) or 2×10⁷ cells/kg(N=2) (see FIGS. 5A and 5B). In some cases, a preconditioningchemotherapeutic treatment of cyclophosphamide (about 2 g/m²),cyclophosphamide (2 g/m²) and etoposide (100 mg/m², administered threetimes daily) or cyclophosphamide (60 mg/kg) and fludarabine (25 mg/m²,administered three to five times daily) was administered to subjectsprior to infusion.

The number of CAR-T cells present in peripheral blood of treatedsubjects was determined post-treatment by performing flow cytometry forsurface expression of EGFRt (CAR-specific marker) and CD4 or CD8.Neurotoxicity also was determined and monitored, and graded based onseverity as described above.

As shown in FIG. 5A, the degree of neurotoxicity observed in this studyin treated subjects correlated to the presence of relatively highertumor burden prior to treatment. As shown in FIG. 5A, subjectsexhibiting severe symptoms of neurotoxicity with a grade of 3 or higheralso exhibited a greater number of CD8+ or CD4+ CAR-T cells inperipheral blood following treatment at the time assessed, indicating acorrelation between tumor burden, the degree of expansion of CAR+ Tcells and neurotoxicity. FIG. 5B shows that subjects determined torequire intensive care unit (ICU) care following treatment were amongthose having relatively higher percentages of bone marrow blasts priorto treatment.

Example 10 Risk-Adapted Dosing of CAR+ T Cells

Five subjects, among those treated as described in Example 9, wereadministered the anti-CD19 CAR-T cells in doses adapted based on tumorburden.

Prior to treatment, tumor burden was assessed by evaluating the percentof marrow blasts present in the bone marrow. Subjects having greaterthan 20% blasts (>20%) in bone marrow were selected for administrationof a dose of CAR-T cells (2×10⁵ T cells/kg) that was relatively lower ascompared to the dose administered to subjects having less than or equalto 20% blasts. Subjects having less than or equal to 20% blasts (≦20%)were selected for administration of a relatively higher dose (2×10⁶CAR-T cells/kg). CAR-expressing T cells were administered to subjects bysingle intravenous (IV) continuous infusion.

At days 0, 1, 3, 7, 10, 14, 21 and 28 post-treatment, the number ofCAR-T cells present in peripheral blood of treated subjects wasdetermined by performing flow cytometry of a cell sample for surfaceexpression of EGFRt (CAR-specific marker) and CD4 or CD8. Subjects alsowere monitored for toxicity, including severe toxic outcomes requiringintensive care unit (ICU) care.

CAR-T cells were observed to expand in all subjects. As compared to agroup of subjects receiving similar treatment, but for which theadministered dosage was not based on tumor burden, the risk-adapteddosing resulted in a reduced incidence of serious toxicity. For example,in subjects for which the administered dosage was not based on tumorburden, seven of eleven subjects were required to have ICU care. Incontrast, none (0%) of the five subjects for which risk-adapted dosingwas used required ICU care.

Example 11 Pre-Conditioning with Fludarabine Prior to CAR-T CellAdministration in Subjects with B Cell Acute Lymphoblastic Leukemia(ALL)

Subjects with CD19⁺ B cell acute lymphoblastic leukemia (ALL) wereadministered 2×10⁶ cells/kg of autologous T cells expressing ananti-CD19 chimeric antigen receptor (CAR). Before treatment, autologousCAR-expressing T cells were generated substantially as described inExample 2.

Prior to administration of the CAR-expressing T cells, subjects weretreated either with 1) 2 g/m² cyclophosphamide (with or withoutadministration of 100 mg/m² etoposide three times daily) (N-=5, no Flutreated group), or were treated with 60 mg/kg (˜2 g/m²) cyclophosphamideand 3 to 5 doses of 25 mg/m² fludarabine (N=10, designated Flu treatedgroup).

At days 0, 1, 3, 7, 10, 14, 21 and 28 post-treatment, the number ofCAR-T cells present in peripheral blood of treated subjects wasdetermined by performing flow cytometry of a cell sample for surfaceexpression of EGFRt (CAR-specific marker) and CD4 or CD8.

As shown in FIGS. 6A and 6B, a greater degree of CAR-T cell expansionand/or persistence, as measured by the presence of CD8⁺ (FIG. 6A) orCD4⁺ (FIG. 6B) CAR-T cells in peripheral blood, was observed from days 7to 28 post-treatment in subjects who were pre-conditioned withcyclophosphamide/fludarabine as compared with those who did not receivefludarabine prior to the administration of CAR-T cells. This resultdemonstrates that pre-conditioning with cyclophosphamide/fludarabine canimpact in vivo CAR-T cell expansion.

As shown in FIG. 6C, a greater percentage of patients exhibitingdisease-free survival over the time period shown was observed amongsubjects having been pre-treated with both cyclophosphamide andfludarabine.

Example 12 Pre-Conditioning with Fludarabine Prior to CAR-T CellAdministration in Subjects with Non-Hodgkin Lymphoma (NHL)

Subjects with Non-Hodgkin Lymphoma (NHL) were administered 2×10⁷cells/kg of autologous T cells expressing an anti-CD19 chimeric antigenreceptor (CAR). Before treatment, autologous CAR-expressing T cells weregenerated substantially as described in Example 2.

Prior to administration of the CAR-expressing T cells, subjects weretreated either with 1) 2-4 g/m² cyclophosphamide (with or withoutadministration of 100-200 mg/m² etoposide three times daily) (N=3,designated no Flu treated group), or 2) with 30-60 mg/kg (˜1-2 g/m²)cyclophosphamide and 3 to 5 doses of 25 mg/m² fludarabine (N=6,designated Cy/Flu treated group).

At days 0, 1, 3, 7, 10, 14, 21 and 28 post-treatment, the number ofCAR-T cells present in peripheral blood of treated subjects wasdetermined by performing flow cytometry of a cell sample for surfaceexpression of EGFRt (CAR-specific marker) and CD4 or CD8.

The results are depicted in FIGS. 7A and 7B, which set forth theCAR-expressing CD8+ or CD4+ T cells, respectively, as total cells per μLor as the total percentage of CD4+ or CD8+ cells, respectively, in thesample. As shown in FIGS. 7A and 7B, a greater degree of CAR-T cellexpansion and/or persistence, as measured by the presence of CD8⁺ (FIG.7A) or CD4⁺ (FIG. 7B) CAR-T cells in peripheral blood followingtreatment, was observed from days 3 to 28 post-treatment in subjects whowere pre-conditioned with cyclophosphamide/fludarabine as compared tothose who did not receive fludarabine prior to the administration ofCAR-T cells. This result demonstrated that pre-conditioning withcyclophosphamide/fludarabine can impact in vivo CAR-T cell expansion insubjects with NHL.

Subjects were monitored for efficacy of treatment by measuring theoverall remission rate (ORR). ORR was determined as the proportion ofsubjects with complete remission (CR) or partial remission (PR), asdetermined by examination of the bone marrow, peripheral blood, andcerebrospinal fluid (CSF), as well as physical examination andevaluation of central nervous system (CNS) symptoms.

The overall remission rate for subjects pre-conditioned withcyclophosphamide/fludarabine was 62% (8/13), including a 38% CR rate and23% PR rate (CR=5/13, PR=3/13), while subjects who did not receivefludarabine pre-conditioning had an overall remission rate of 50%(6/12), including an 8% CR rate and a 42% PR rate (CR=1/12, PR=5/12).

The present invention is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the invention. Various modifications tothe compositions and methods described will become apparent from thedescription and teachings herein. Such variations may be practicedwithout departing from the true scope and spirit of the disclosure andare intended to fall within the scope of the present disclosure.

1-76. (canceled)
 77. A method of treatment, comprising: administering aconsecutive dose of cells expressing a chimeric antigen receptor (CAR)to a subject having a disease or condition and having been previouslyadministered a first dose of cells expressing a CAR, said first dosecomprising no more than about 1×10⁶ of the cells per kilogram bodyweight of the subject, no more than about 1×10⁸ of the cells, and/or nomore than about 1×10⁸ of the cells/m² of the subject, wherein: the CARexpressed by the cells in the first dose and the CAR expressed by thecells of the consecutive dose each, individually, binds to an antigenexpressed by a cell or tissue of the disease or condition or associatedwith the disease or condition; and the consecutive dose of cells isadministered at a point in time that is at least or more than about 14days after and less than about 28 days after initiation of theadministration of the first dose of cells.
 78. The method of treatmentof claim 77, further comprising administering to the subject the firstdose of cells.
 79. The method of claim 77, wherein, at the time of theadministration of the consecutive dose: (i) the serum level in thesubject of a factor indicative of cytokine release syndrome (CRS) is nomore than about 10 times the amount, no more than about 25 times, and/orno more than about 50 times that in the subject immediately prior to theadministration of the first dose; and/or (ii) the subject does notexhibit grade 3 or higher neurotoxicity; and/or (iii) a CRS-relatedoutcome or symptom of neurotoxicity in the subject has reached a peaklevel and begun to decline following the administration of the firstdose; and/or (iv) the subject does not exhibit a detectable humoral orcell-mediated immune response against the CAR expressed by the cells ofsaid first dose.
 80. The method of claim 77, wherein prior to theadministration of the first dose of cells, said subject had not receiveda dose of cells expressing the CAR expressed by the cells in the firstdose.
 81. The method of claim 77, wherein the antigen bound by the CARexpressed by the cells in the first dose and the antigen bound by theCAR expressed by the cells in the consecutive dose is the same antigenand/or the CAR expressed by the cells in the first dose and the CARexpressed by the cells in the consecutive dose contain the sameantigen-binding domain.
 82. The method of claim 77, wherein: the CARexpressed by the cells in the consecutive dose contains at least oneimmunoreactive epitope present in the CAR expressed by the cells in thefirst dose; or the CAR expressed by the cells in the consecutive dose isidentical to the CAR expressed by the cells in the first dose or issubstantially identical to the CAR expressed by the cells in the firstdose.
 83. The method of claim 77, wherein the disease or condition is atumor or cancer.
 84. The method of claim 77, wherein: the first and/orconsecutive dose of cells comprises cells in an amount sufficient forreduction in burden of the disease or condition in the subject; and/orthe administration of the consecutive dose leads to a reduction inburden of the disease or condition in the subject.
 85. The method ofclaim 78, wherein the method reduces burden of the disease or conditionto a greater degree and/or for a greater period of time as compared to amethod comprising an alternative dosing regimen wherein the subject isadministered the first dose of cells and the second dose of cells in asingle dose.
 86. The method of claim 77, wherein (i) the maximum numberof CAR-expressing cells, (ii) the area under the curve (AUC) for bloodconcentration of CAR-expressing cells over time beginning immediatelyafter administration of the first dose and/or (iii) the duration ofdetectable CAR-expressing cells in the subject following saidadministration of the consecutive dose of cells is greater, as comparedto that achieved via a method comprising an alternative dosing regimenwherein the subject is administered the first dose of cells and thesecond dose of cells as a single dose.
 87. The method of claim 77,wherein: the maximum concentration or number of CAR-expressing cells inthe blood of the subject is at least at or about 10 CAR-expressing cellsper microliter, at least 50% of the total number of peripheral bloodmononuclear cells (PBMCs), at least at least about 1×10⁵ CAR-expressingcells, or at least 5,000 copies of CAR-encoding DNA per micrograms DNA;and/or at day 90 following the initiation of the administration of thefirst dose of cells, CAR-expressing cells are detectable in the blood orserum of the subject; and/or at day 90 following the initiation of theadministration of the first dose of cells, the blood of the subjectcontains at least 20% CAR-expressing cells, at least 10 CAR-expressingcells per microliter or at least 1×10⁴ CAR-expressing cells.
 88. Themethod of claim 77, further comprising: i) assessing a factor indicativeof disease after administration of the first dose of cells and prior toadministration of the consecutive dose of cells; ii) based on the resultof the assessment, determining the consecutive dose of cells to beadministered to the subject, and iii) if the assessment determines thatthe subject has morphologic disease, administering to the subject aconsecutive dose comprising less than or about the same number ofCAR-expressing cells as the number of CAR-expressing cells in the firstdose; and/or if the assessment determines that the subject has minimalresidual disease, administering to the subject a consecutive dosecomprising an increased number of CAR-expressing cells as compared tothe first dose.
 89. The method of claim 77, wherein: if at a time justprior to initiation of administration of the consecutive dose of cellsthe subject exhibits or is suspected of exhibiting morphologic disease,the consecutive dose comprises less than or about the same number ofCAR-expressing cells as the number of CAR-expressing cells in the firstdose; and if at a time just prior to initiation of administration of theconsecutive dose of cells the subject exhibits or is suspected ofexhibiting minimum residual disease, the consecutive dose comprises anincreased number of CAR-expressing cells as compared to the first dose.90. The method of claim 77, wherein the consecutive dose comprises anincreased number of CAR-expressing cells as compared to the first dose.91. The method of claim 77, wherein the number of CAR-expressing cellsadministered in the consecutive dose comprises between about 2×10⁶ perkilogram (kg) body weight and about 6×10⁶/kg, between about 2.5×10⁶/kgand about 5.0×10⁶/kg, or between about 3.0×10⁶/kg and about 4.0×10⁶/kg,each inclusive.
 92. The method of claim 77, wherein the time between thefirst and consecutive dose is from 15 to 27 days, inclusive.
 93. Themethod of claim 77, further comprising administering a chemotherapeuticagent prior to the administration of the consecutive dose of cells. 94.The method of claim 77, wherein the subject has been previously treatedwith a chemotherapeutic agent prior to administration of the first doseand/or prior to the administration of the consecutive dose.
 95. Themethod of claim 93, wherein the chemotherapeutic agent comprises anagent selected from the group consisting of cyclophosphamide andfludarabine.
 96. The method of claim 93, wherein the chemotherapeuticagent comprises a combination of cyclophosphamide and fludarabine. 97.The method of claim 94, wherein the chemotherapeutic agent comprises anagent selected from the group consisting of cyclophosphamide andfludarabine.
 98. The method of claim 94, wherein the chemotherapeuticagent comprises a combination of cyclophosphamide and fludarabine. 99.The method of claim 77, wherein the subject has been previously treatedwith a chemotherapeutic agent prior to the initiation of administrationof the first dose and/or wherein the subject has been previously treatedwith a chemotherapeutic agent subsequently to the initiation of theadministration of the first dose, and prior to the initiation ofadministration of the consecutive dose.
 100. The method of claim 77,wherein: the first dose is a split dose, wherein the cells of the firstdose have been administered in a plurality of compositions, collectivelycomprising the cells expressing the CAR of the first dose, over a periodof no more than three days; and/or the consecutive dose is a split dose,wherein the cells of the consecutive dose are administered in aplurality of compositions, collectively comprising the cells of theconsecutive dose, over a period of no more than three days.
 101. Themethod of claim 78, further comprising administering a chemotherapeuticagent prior to the administration of the first dose of cells.
 102. Themethod of claim 77, wherein the disease or condition is a leukemia orlymphoma.
 103. The method of claim 77, wherein the disease or conditionis acute lymphoblastic leukemia or non-Hodgkin lymphoma (NHL).
 104. Themethod of claim 77, wherein the number of CAR⁺ cells administered in thefirst dose is at or about or no more than at or about 1×10⁶ per kilogramof the subject and/or the number of CAR⁺ cells administered in theconsecutive dose is at or about 3×10⁶ per kilogram of the subject. 105.The method of claim 77, further comprising administering to the subjectone or more additional subsequent doses, wherein the first of said oneor more additional subsequent doses is administered at a time that is atleast or greater than 14 days after the initiation of the administrationof the consecutive dose.
 106. The method of claim 77, wherein the cellsare T cells.
 107. A method of treatment, comprising administering aconsecutive dose of cells expressing a chimeric antigen receptor (CAR)to a subject having a disease or condition and having been previouslyadministered a first dose of cells expressing a CAR, said first dosecomprising the cells in an amount sufficient to reduce burden of thedisease or condition in the subject, wherein: the CAR expressed by thecells in the first dose and the CAR expressed by the cells of theconsecutive dose each, individually, binds to an antigen expressed by acell or tissue of the disease or condition or associated with thedisease or condition; and the consecutive dose is administered at apoint in time at which: (i) a clinical risk for neurotoxicity,cytokine-release syndrome (CRS), macrophage activation syndrome, ortumor lysis syndrome, is not present or has passed or has subsidedfollowing said administration of the first dose, (ii) a biochemicalreadout evidencing CRS, neurotoxicity, macrophage activation syndrome,or tumor lysis syndrome, is not present or has passed or has subsidedfollowing said administration of the first dose, iii) a CRS-relatedoutcome in the subject has reached a peak level and begun to declinefollowing administration of the first dose, and/or (iv) a serum level ofa factor indicative of cytokine-release syndrome (CRS) or neurotoxicityin the subject is no more than about 10 times, no more than about 25times, and/or no more than about 50 times the serum level of saidindicator in the subject immediately prior to said administration of thefirst dose; and the subject does not exhibit a detectable adaptive hostimmune response specific for the CAR expressed by the cells of saidfirst dose.
 108. The method of treatment of claim 107, comprisingadministering to the subject the first dose of cells.
 109. The method ofclaim 108, wherein: (i) the administration of the first dose of cellsdoes not induce severe CRS in the subject or does not induce CRS in thesubject; (ii) the administration of the first dose of cells does notinduce grade 3 or higher neurotoxicity in the subject; (iii) based onclinical data, the administration of the first dose of cells does notinduce severe CRS in a majority of subjects so-treated; and/or (iv)based on clinical data, the administration of the first dose of cellsdoes not induce grade 3 or higher neurotoxicity in a majority ofsubjects so-treated.
 110. The method of claim 107, wherein: theconsecutive dose of cells is administered at a time point that isbetween about 9 and about 35 days, between about 14 and about 28 days,between 15 and 27 days or is between 17 days and about 21 days, eachinclusive, after initiation of the administration of the first dose ofcells; and/or said first dose comprises no more than about 1×10⁶ of thecells per kilogram body weight of the subject, no more than about 1×10⁸of the cells, or no more than about 1×10⁸ of the cells/m² of thesubject.
 111. The method of claim 107, wherein the CRS-related outcomeis selected from the group consisting of fever, hypotension, hypoxia,neurologic disturbances, and a serum level of an inflammatory cytokineor C reactive protein (CRP).
 112. The method of 107, wherein: thedisease is a cancer and the subject does not exhibit morphologic diseaseat a time just prior to initiation of the administration of theconsecutive dose of cells; and/or the disease is a leukemia or lymphomaand the subject does not exhibit greater than 5% blast cells in the bonemarrow at a time just prior to the administration of the consecutivedose of cells.
 113. The method of claim 112, wherein the subjectexhibits detectable molecular disease and/or minimum residual disease ata time just prior to the administration of the consecutive dose ofcells.
 114. The method of claim 107, wherein the number ofCAR-expressing cells administered in the first dose is between about0.5×10⁶/kg body weight of the subject and 3×10⁶/kg, between about0.75×10⁶/kg and 2.5×10⁶/kg or between about 1×10⁶/kg and 2×10⁶/kg, eachinclusive.
 115. The method of claim 107, wherein the number ofCAR-expressing cells in the consecutive dose comprises between about2×10⁶ per kilogram (kg) body weight and about 6×10⁶/kg, between about2.5×10⁶/kg and about 5.0×10⁶/kg, or between about 3.0×10⁶/kg and about4.0×10⁶/kg, each inclusive.
 116. A method of treatment, comprisingadministering a consecutive dose of cells expressing a chimeric antigenreceptor (CAR) to a subject having a disease or condition previouslyadministered a first dose of cells expressing a CAR, wherein: the CARexpressed by the cells in the first dose and the CAR expressed by thecells of the consecutive dose each, individually, binds to an antigenexpressed by a cell or tissue of the disease or condition or associatedwith the disease or condition; and the consecutive dose of cells isadministered at a time point that is at least or more than about 14 daysafter and less than about 28 days after initiation of the first dose.117. The method of claim 116, wherein the number of CAR-expressing cellsadministered in the consecutive dose is increased as compared to thefirst dose.
 118. The method of claim 117, wherein the number of CAR⁺cells per kilogram administered in the consecutive dose is at least ator about 2 times or at or about 3 times greater than the number of CAR⁺cells per kilogram administered in the first dose.
 119. An article ofmanufacture, comprising: a plurality of sealable containers, eachindividually comprising a unit dose of cells expressing a chimericantigen receptor (CAR) for administration to a subject, said unit dosecomprising about 1×10⁸ of the cells, no more than about 1×10⁸ of thecells, about 5×10⁷ of the cells, no more than about 5×10⁷ of the cells,about 1×10⁶ cells per kg of the subject, or no more than about 1×10⁶ ofthe cells per kg of the subject; packaging material; and a label orpackage insert comprising instructions for administering a plurality ofsaid unit doses to the subject by carrying out a first administrationand a consecutive administration, said first administration comprisingdelivering one of said unit doses to the subject and said consecutiveadministration comprising administering one or a plurality of said unitdoses to the subject.